Engineered tils expressing secreted il-2 fusion proteins and uses thereof

Abstract

The present application relates to a kind of expression secretory IL-2 fusion protein of engineered TIL cell and its application.It is specifically, the present application provides the fusion protein of IL-2 and CD25, and is designed using the double hypoxia response element (HRE) and oxygen-dependent degradation domain (ODD) element group hypoxia sensing safety switch, control fusion protein only expresses in hypoxic environment, on the one hand, realizes the specific concentration expression of IL-2 in tumor microenvironment, enhances tumor local concentration, can effectively maintain the anti-tumor activity of TIL in tumor tissue;On the other hand, the security risk brought by IL-2 system exposure is minimized.Meanwhile, the endogenous TIM-3 expression of TIL cell is silenced, so as to effectively reduce TIL exhaustion, further enhance the anti-tumor activity of TIL cell.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and cell therapy, in particular to engineered TILs expressing secreted IL-2 fusion proteins and applications thereof. BACKGROUND

[0002] Tumor infiltrating lymphocytes (TILs) are a highly heterogeneous subset of lymphocytes present in the tumor microenvironment (TME), mainly including cytotoxic T cells, helper T cells, regulatory T cells, B cells, NK cells, etc. On February 16, 2024, the U.S. FDA accelerated the approval of Amtagvi (lifileucel) developed by Iovance Biotherapeutics for the treatment of unresectable / metastatic melanoma that has progressed after prior PD-1 therapy. Amtagvi (lifileucel), as the first FDA-approved TIL cell product, is an innovative individualized cancer immunotherapy. In addition to melanoma, in recent years, TIL cell therapy has also shown significant efficacy in lung cancer, cervical cancer, endometrial cancer, ovarian cancer, colon cancer, head and neck cancer, and other solid tumors.

[0003] However, severe treatment-related toxicities have always been the main limiting factor for the clinical application and promotion of TIL cell therapy. Generally, toxicities associated with TIL cell administration are less common and manifest as temporary symptoms such as dyspnea, fever, chills, tachycardia, or hypertension, which can often be managed with basic supportive measures. After TIL infusion, patients will receive high-dose IL-2 treatment, and high-dose IL-2 stimulation has long been an important component of TIL cell therapy. IL-2 is an important immunomodulatory factor that can promote the growth and activity of TIL cells. Through IL-2 treatment, the attack power of TIL cells on cancer cells can be further enhanced [Ann Oncol. 2019; 30(12): 1902-1913]. However, the toxicity from high-dose IL-2 is very severe and is usually manifested in multiple organ systems. Systemic capillary leak syndrome, pulmonary edema, and renal failure are common toxic reactions that require monitoring and care in the intensive care unit (ICU). Currently, the severe toxicity of supportive infusion of high-dose IL-2 is the main reason for TIL cell therapy to be performed only in specialized centers with experienced staff.

[0004] To date, next-generation tumor-infiltrating lymphocyte (TIL) attempts in clinical applications have mainly focused on engineering TILs to overexpress cytokines such as IL-2, IL-12, IL-7, and IL-15. In a phase I / II trial, after inserting IL-2 into TILs, the infusion was supported in vitro by removing IL-2, and in patients treated with IL-2 transduced TILs, only 2 / 12 (17%) observed objective response rate (ORR), while the ORR of patients treated with unmodified TILs plus high-dose IL-2 was about 50% [Hum Gene Ther. 2008; 19(5):496-510.]. Another phase I trial used TILs expressing IL-12 under the control of an activated T cell nuclear factor (NFAT)-inducible promoter, which showed good clinical efficacy in metastatic melanoma: patients receiving 0.3 to 300 million NFAT-IL-12 engineered TILs achieved an ORR of 63%, which was 10 to 100 times less than the number of cells used in traditional TIL therapy; however, unexpectedly high levels of IL-12 and IFN-γ in serum and significant toxicity were also observed [Clin Cancer Res. 2015; 21(10):2278-88].

[0005] On the other hand, anti-PD-1 / PD-L1 antibodies have far surpassed anti-CTLA-4 antibodies in clinical applications due to their better clinical efficacy and tolerability. Nevertheless, a large proportion of patients do not respond to ICIs treatment, and some patients experience tumor regression, develop resistance to ICIs, and relapse, and only a small proportion of patients achieve durable efficacy [Annu Rev Pathol. 2021; 16:223-249]. In the observation of PD-1 resistant patients, it was found that multiple emerging alternative immune checkpoints, including TIM-3, were compensatorily upregulated, and when a TIM-3 combined with PD-1 treatment regimen was used, the responsiveness of anti-PD-1 treatment was restored [Br J Cancer. 2021; 125(7):927-938]. Similar to PD-1, TIM-3 is also highly expressed on exhausted T cells and NK cells in chronic infections and cancers. High expression of TIM-3 on CD8+ T cells and NK cells is associated with an exhausted phenotype, while expression of TIM-3 on tumor-associated FoxP3+ regulatory T cells (Tregs) marks a subset of Tregs with stronger suppressive function and recovery capacity.

[0006] Therefore, in order to reduce the toxicity associated with high-dose IL-2 clinical applications and improve the anti-tumor activity of TIL cells and reduce TIL cell exhaustion, there is an urgent need in the art to develop safe and controllable IL-2 secreting functional enhanced TIL cells. SUMMARY

[0007] The object of the present application is to provide a functional enhanced TIL cell with safe and controllable secretion of IL-2 and its application in tumor treatment.

[0008] In the first aspect of the present application, an engineered TIL cell is provided, which has the following characteristics:

[0009] (a) the TIL cell expresses an exogenous secreted IL-2 fusion protein in a hypoxic environment under the regulation of a promoter comprising a hypoxia response element (HRE); and

[0010] (b) the endogenous TIM-3 expression of the TIL cell is silenced;

[0011] wherein the structural formula of the secreted IL-2 fusion protein is shown as formula (I):

[0012] Z1-L1-Z2-L2-Y (I)

[0013] wherein,

[0014] each of “-” is independently nothing or a connecting peptide;

[0015] Z1 is an IL-2 element;

[0016] each of L1 and L2 is independently nothing or a linker;

[0017] Z2 is a CD25 element;

[0018] Y is an oxygen-dependent degradation domain (ODD) element.

[0019] In another preferred embodiment, the IL-2 element comprises a full-length, mature form of IL-2 or an active fragment thereof.

[0020] In another preferred embodiment, the IL-2 comprises a wild type and a mutant.

[0021] In another preferred embodiment, the amino acid sequence of the IL-2 element is shown as SEQ ID NO: 1.

[0022] In another preferred embodiment, the CD25 element is an extracellular region of interleukin 2 receptor alpha subunit.

[0023] In another preferred embodiment, the amino acid sequence of the CD25 element is shown as SEQ ID NO: 2.

[0024] In another preferred embodiment, the ODD element is an oxygen-dependent degradation domain of HIF-1α subunit.

[0025] In another preferred embodiment, the amino acid sequence of the ODD element is set forth in SEQ ID NO: 3.

[0026] In another preferred embodiment, the linker is a non-cleavable flexible linker.

[0027] In another preferred embodiment, the amino acid sequence of the linker is set forth in SEQ ID NO: 4.

[0028] In another preferred embodiment, the amino acid sequence of the IL-2 element is set forth in SEQ ID NO: 1; the CD25 element is an extracellular region of interleukin 2 receptor alpha subunit, the amino acid sequence of which is set forth in SEQ ID NO: 2; and the ODD element is an oxygen-dependent degradation domain of HIF-1 alpha subunit, the amino acid sequence of which is set forth in SEQ ID NO: 3.

[0029] In another preferred embodiment, the amino acid sequence of the secreted IL-2 fusion protein is set forth in SEQ ID NO: 5.

[0030] In another preferred embodiment, the engineered TIL cell contains a nucleic acid construct, wherein the first expression cassette of the nucleic acid construct comprises a coding sequence of the secreted IL-2 fusion protein.

[0031] In another preferred embodiment, the first expression cassette comprises a first promoter, wherein the first promoter is an inducible promoter, and the inducible promoter is a promoter comprising hypoxia response elements (HREs).

[0032] In another preferred embodiment, the inducible promoter is a promoter comprising n hypoxia response elements (HREs), i.e., a promoter comprising n x HRE elements, wherein n is an integer selected from 3-12, and preferably n is 9.

[0033] In another preferred embodiment, the inducible promoter is an IL-12-mini promoter comprising 9 x HRE elements.

[0034] In another preferred embodiment, the nucleotide sequence of the IL-12-mini promoter comprising 9 x HRE elements is set forth in SEQ ID NO: 9.

[0035] In another preferred embodiment, the first expression cassette has the following structure set forth in Formula (II) from 5’ end to 3’ end:

[0036] P1-SP-Z1’-L1’-Z2’-L2’-Y’ (II)

[0037] wherein,

[0038] each of “-” is independently a linkage;

[0039] P1 is a first promoter;

[0040] SP is a coding sequence of a signal peptide;

[0041] Z1' is a coding sequence of an IL-2 element;

[0042] L1' and L2' are each independently a coding sequence of a linker;

[0043] Z2' is a coding sequence of a CD25 element;

[0044] Y' is a coding sequence of an oxygen-dependent degradation domain (ODD) element.

[0045] In another preferred embodiment, the nucleic acid construct further comprises a second expression cassette comprising a coding sequence of a shRNA targeting an endogenous TIM-3 transcript.

[0046] In another preferred embodiment, the nucleotide sequence of the shRNA is set forth in SEQ ID NO: 6.

[0047] In another preferred embodiment, the second expression cassette has the following structure set forth in formula (III) from 5' end to 3' end:

[0048] P2-R (III)

[0049] wherein "-" is a linking bond;

[0050] P2 is a second promoter;

[0051] R is a coding sequence of a shRNA targeting an endogenous TIM-3 transcript.

[0052] In another preferred embodiment, the second promoter is a U6 promoter.

[0053] In another preferred embodiment, the nucleic acid construct has the following structure set forth in formula (IV) from 5' end to 3' end:

[0054] P2-R-P1-SP-Z1'-L1'-Z2'-L2'-Y' (IV)

[0055] wherein each element is as described above.

[0056] In a second aspect of the present application, there is provided an expression vector comprising a nucleic acid construct as set forth in formula (IV):

[0057] P2-R-P1-SP-Z1'-L1'-Z2'-L2'-Y' (IV)

[0058] wherein,

[0059] each - is independently a linker;

[0060] P2 is a second promoter;

[0061] R is a coding sequence of a shRNA targeting an endogenous TIM-3 transcript;

[0062] P1 is a first promoter;

[0063] SP is a coding sequence of a signal peptide;

[0064] Z1’ is a coding sequence of an IL-2 element;

[0065] L1’ and L2’ are each independently a coding sequence of no or a linker;

[0066] Z2’ is a coding sequence of a CD25 element;

[0067] Y’ is a coding sequence of an oxygen-dependent degradation domain (ODD) element.

[0068] In another preferred embodiment, the amino acid sequence of the IL-2 element is set forth in SEQ ID NO: 1.

[0069] In another preferred embodiment, the CD25 element is an extracellular region of an interleukin 2 receptor alpha subunit.

[0070] In another preferred embodiment, the amino acid sequence of the CD25 element is set forth in SEQ ID NO: 2.

[0071] In another preferred embodiment, the ODD element is an oxygen-dependent degradation domain of a HIF-1 alpha subunit.

[0072] In another preferred embodiment, the amino acid sequence of the ODD element is set forth in SEQ ID NO: 3.

[0073] In another preferred embodiment, the linker is a non-cleavable flexible linker.

[0074] In another preferred embodiment, the amino acid sequence of the linker is set forth in SEQ ID NO: 4.

[0075] In another preferred embodiment, the first promoter is an inducible promoter, and the inducible promoter is a promoter comprising hypoxia response elements HREs.

[0076] In another preferred embodiment, the inducible promoter is a promoter comprising n hypoxia response elements HREs, i.e. a promoter comprising n x HRE elements, n is an integer selected from 3-12, preferably n is 9.

[0077] In another preferred embodiment, the inducible promoter is an IL-12-mini promoter containing 9xHRE elements.

[0078] In another preferred embodiment, the nucleotide sequence of the IL-12-mini promoter containing 9xHRE elements is shown as SEQ ID NO: 9.

[0079] In another preferred embodiment, the second promoter is a U6 promoter.

[0080] In another preferred embodiment, the nucleotide sequence of the shRNA is shown as SEQ ID NO: 6.

[0081] In another preferred embodiment, the vector includes a plasmid, a viral vector (e.g., a lentiviral vector).

[0082] In another preferred embodiment, the vector is a pCDH series lentiviral vector.

[0083] In a third aspect of the present application, a method for preparing the engineered TIL cell as described in the first aspect of the present application is provided, comprising the following steps:

[0084] (1) providing a TIL cell to be modified; and

[0085] (2) modifying the TIL cell so that the endogenous TIM-3 expression of the TIL cell is silenced and the TIL cell contains a coding gene of the secreted IL-2 fusion protein, thereby obtaining the engineered TIL cell.

[0086] In another preferred embodiment, in step (2), the following steps are included:

[0087] (2a) introducing a first expression cassette containing a coding gene of the secreted IL-2 fusion protein into the TIL cell;

[0088] (2b) introducing a second expression cassette containing a coding sequence of an shRNA targeting an endogenous TIM-3 transcript into the TIL cell.

[0089] In another preferred embodiment, the steps (2a) and (2b) can be performed in any order or simultaneously.

[0090] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0091] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same vector.

[0092] In another preferred embodiment, the vector includes a plasmid, a viral vector (e.g., a lentiviral vector).

[0093] In another preferred embodiment, the vector is a pCDH series lentivirus vector.

[0094] In another preferred embodiment, the step (2) comprises introducing the expression vector of the second aspect of the present application into the TIL cell, thereby obtaining the engineered TIL cell.

[0095] In a fourth aspect of the present application, there is provided a cell preparation comprising the engineered TIL cell of the first aspect of the present application, and a pharmaceutically acceptable carrier, diluent or excipient.

[0096] In another preferred embodiment, the cell preparation is for preventing and / or treating a tumor.

[0097] In another preferred embodiment, the cell preparation is a liquid preparation.

[0098] In another preferred embodiment, the cell preparation is in the form of an injection.

[0099] In another preferred embodiment, the cell preparation comprises a therapeutically effective amount of the engineered TIL cell of the first aspect of the present application.

[0100] In another preferred embodiment, the concentration of the engineered TIL cell in the cell preparation is 1 x 10 3 - 1 x 10 8 cells / ml, preferably 1 x 10 4 - 1 x 10 7 cells / ml.

[0101] In another preferred embodiment, the cell preparation further comprises a second active ingredient against a tumor, preferably a second antibody or a chemotherapeutic agent.

[0102] In a fifth aspect of the present application, there is provided use of the engineered TIL cell of the first aspect of the present application in the manufacture of a medicament or preparation for preventing and / or treating a tumor.

[0103] In another preferred embodiment, the tumor is a solid tumor.

[0104] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, cervical cancer, melanoma, colorectal cancer, head and neck cancer, liver cancer, ovarian cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, prostate cancer, esophageal cancer, and breast cancer.

[0105] In a sixth aspect of the present application, there is provided a method for preventing and / or treating a tumor, the method comprising administering to a subject in need thereof the engineered TIL cell of the first aspect of the present application, or the cell preparation of the fourth aspect of the present application.

[0106] In another preferred embodiment, the subject in need thereof comprises a human or a non-human mammal.

[0107] In another preferred embodiment, the tumor is a solid tumor.

[0108] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, cervical cancer, melanoma, colorectal cancer, head and neck cancer, liver cancer, ovarian cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, prostate cancer, esophageal cancer, and breast cancer.

[0109] It should be understood that, within the scope of the present application, each of the technical features described above and the technical features specifically described below (in the examples, for example) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one. BRIEF DESCRIPTION OF DRAWINGS

[0110] Figure 1 The structural schematic diagram of the nucleic acid construct expressed in 1#TIL, 2#TIL, 3#TIL, 4#TIL and 5#TIL, respectively, is shown.

[0111] Figure 2 The results of ELISA detection of the amount of IL-2 secreted by the TIL cells in each group under normal culture conditions (A) and hypoxic culture conditions (B) are shown.

[0112] Figure 3 The TIM-3 flow detection results are shown.

[0113] Figure 4 The proportions of Treg, CD4+T and CD8+T cells in 1#TIL and Ctrl TIL cells are shown, wherein A shows the proportions of CD4+T and CD8+T cells in Ctrl TIL cells; B shows the proportions of CD4+T and CD8+T cells in 1#TIL cells; C shows the proportion of FOXP3+CD25+T cells in CD4+T cells in Ctrl TIL cells, and CD4+FOXP3+CD25+T is the phenotypic characteristic of Treg cells, i.e., the figure shows the proportion of Treg cells in Ctrl TIL cells; and D shows the proportion of Treg cells in Ctrl TIL cells.

[0114] Figure 5 The killing ability of 1#TIL, 2#TIL, 3#TIL and Ctrl TIL on tumor cells is shown. DETAILED DESCRIPTION

[0115] Through extensive and in-depth research, the present inventors first provide a secreted IL-2 fusion protein with a specific structure, which mainly comprises an IL-2 element and a CD25 active fragment. In addition, the fusion protein of the present application adopts a double hypoxia-inducible safety switch design to control the expression of the fusion protein only in a hypoxic environment, on the one hand, realizing the specific concentration expression of IL-2 in the tumor microenvironment, enhancing the local concentration in the tumor, and effectively promoting the growth of TILs in the tumor tissue; on the other hand, the safety hazards brought by systemic IL-2 exposure are minimized, and the safety of clinical treatment is improved. Specifically, the double hypoxia-inducible switch design is achieved by appending an oxygen-dependent degradation domain (ODD) of 203 amino acids at the carboxy terminus of hypoxia-inducible factor HIF-1a to the IL-2 fusion protein, and modifying the promoter of the IL-2 fusion protein in the long terminal repeat (LTR) enhancer region of the vector, so as to contain a series of 9 consecutive hypoxia response elements (9xHRE), thereby allowing HIF1a-mediated transcription of the IL-2 fusion protein. The double hypoxia-inducible switch design is superior to the single hypoxia-inducible switch design, and the latter shows the inaccuracy of the expression of the IL-2 fusion protein under normoxic conditions, while the former strictly limits the expression of the IL-2 fusion protein in a hypoxic environment.

[0116] In order to further reduce the exhaustion of TIL cells, TIM-3 shRNA is also introduced, which can effectively knock down the expression level of TIM-3 in TIL cells, intervene in the T cell response inhibition and functional impairment mediated by the interaction between TIM-3 and its ligand (mainly Galectin-9), and then improve the exhaustion state of T cells.

[0117] On this basis, the present application is completed.

[0118] Terms

[0119] In order to facilitate the understanding of the present disclosure, first define certain terms. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0120] The term "about" can refer to a value or composition within an acceptable error range of a specific value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0121] The term "administration" refers to the physical introduction of the products of the present application into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0122] It should be understood that the amino acid names herein are in the internationally accepted one-letter code, and the corresponding amino acid names in the three-letter code are as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gin (Q), Glu (E), Gly (G), His (H), lie (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0123] IL-2

[0124] Interleukin 2 (IL-2) has a molecular weight of 15 kD, is a glycoprotein containing 113 amino acids, and is mainly synthesized by T cells (especially CD4+ T cells) after being stimulated by antigens or mitogens; B cells, NK cells and monocyte-macrophages can also produce IL-2. IL-2 can promote T lymphocyte proliferation and differentiation, induce killer cells with cytotoxic activity, induce and enhance the activity of killer T cells, monocytes and macrophages, etc. After TIL infusion, the patient will receive high-dose IL-2 treatment, and high-dose IL-2 stimulation has been an important part of TIL cell therapy for a long time. IL-2 is an important immunomodulatory factor that can promote the growth and activity of TIL cells. Through IL-2 treatment, the attack power of TIL cells on cancer cells can be further enhanced. However, the toxicity from high-dose IL-2 is very serious and is usually manifested in multiple organ systems. Systemic capillary leak syndrome, pulmonary edema and renal failure are common toxic reactions that require monitoring and care in the intensive care unit (ICU).

[0125] In the present application, IL-2 is fused and expressed with a CD25 active fragment, and the expression of the fusion protein is induced and started under the condition of low oxygen in the tumor microenvironment, so that IL-2 is enriched in the tumor and maintains a high concentration in the local tumor, effectively promoting the growth of TIL in the tumor tissue, while reducing the side effects of high-dose IL-2 on normal tissues.

[0126] The IL-2 used in the present application includes full-length, mature form IL-2, or an active fragment thereof, and the IL-2 includes wild type and mutant.

[0127] In a preferred example, the amino acid sequence of the IL-2 is as follows:

[0128] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL (SEQ ID NO: 1).

[0129] CD25

[0130] The interleukin-2 receptor (IL-2R) is composed of three subunits, a (IL-2Rα, CD25), β (IL-2Rβ, CD122) and γ common (IL-2Rγ, CD132). CD25 is the only subunit that binds IL-2. IL-2 is a cytokine with low affinity, which first binds to CD25 (IL-2Rα) to form a low-affinity complex, and this binding promotes the formation of a heterodimer of IL-2Rβ and IL-2Rγ, thereby forming a high-affinity IL-2R complex, thereby promoting the formation of IL2Rα / β / γ and IL-2 tetramer, and finally initiating signal transduction. The high-affinity IL-2R is mainly expressed on Treg cells, and the CD25 molecule is also considered to be a characteristic marker molecule on the surface of regulatory T cells. The medium-affinity IL-2R is composed of two subunits (CD122 and CD132), which is mainly expressed on the surface of CD8-positive memory T cells and most NK cells, and the heterodimer of IL2Rβ and IL2Rγ binds IL-2 to independently initiate signal transduction, and IL2Rα and IL-2 alone do not have signal transduction function.

[0131] In the present application, IL-2 is expressed in fusion with CD25, wherein IL-2 and CD25 are connected by a non-cleavable linker. Due to the increase in molecular weight, this non-cleavable IL-2 / CD25 fusion protein increases the half-life of IL-2; on the other hand, this design eliminates the role of cell surface CD25 in IL-2R signal transduction, thereby selectively promoting the activation and expansion of CD8+ T cells and NK cells, reducing IL-2-induced Treg expansion, thereby reducing the immunosuppressive effect of the tumor microenvironment, and further enhancing the tumor-killing activity of TIL.

[0132] The CD25 used in the present application includes full-length CD25 or an active fragment thereof, and the CD25 includes wild type and mutant.

[0133] In a preferred embodiment, the CD25 is an extracellular region active fragment of CD25, and the specific amino acid sequence is as follows:

[0134] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ (SEQ ID NO: 2).

[0135] Oxygen-dependent degradation domain of HIF-1a

[0136] Hypoxia is a common feature of solid tumors, where high proliferation and metabolic demands of tumor cells, coupled with inefficient tumor vasculature, result in insufficient oxygen supply (<2% O2) compared to healthy organs / tissues (5-10% O2). This feature of hypoxia is closely associated with tumor proliferation, differentiation, angiogenesis, energy metabolism, and cancer drug resistance, poor patient prognosis. Because hypoxia can distinguish the tumor microenvironment (TME) from healthy, normally oxygenated tissues, it is an ideal marker.

[0137] When the oxygen content decreases in growing tumors, hypoxia activates a gene called HIF1 (hypoxia-inducible factor) and reduces or even shuts down oxygen-consuming processes in mitochondria, especially oxidative phosphorylation, by acting as a transcriptional regulator, thus making glycolysis the main energy production method for cancer cells. HIF1 is a heterodimer composed of an oxygen-sensitive subunit, HIF-1a, and a constitutively expressed HIF-1b. Under hypoxic conditions, HIF-1a is degraded, but under hypoxic conditions, HIF-1a stably forms a heterodimer with HIF-1b.

[0138] The oxygen-dependent degradation domain (ODD) of HIF-1a is the key site of oxygen regulation of HIF1, and under normal oxygen saturation, HIF-1a is rapidly degraded by the ubiquitin proteasome pathway mediated by ODD; ODD fusion expressed protein, under normoxic conditions, ODD is ubiquitinated, leading to protein degradation by ubiquitin proteasome, but under hypoxic conditions, the level of ubiquitination and hydroxylation decreases, and the degradation of HIF-1a is inhibited.

[0139] In the present application, the IL-2 / CD25 fusion protein is fused with ODD for expression. Meanwhile, the promoter of IL-2 in the long terminal repeat (LTR) enhancer region of the vector is modified to contain a series of consecutive hypoxia response elements (HREs), thereby allowing HIF-1α-mediated IL-2 transcription. The design of this dual hypoxia sensing system ensures that IL-2 is only expressed in a hypoxic environment, thereby achieving specific and concentrated expression of IL-2 in the tumor microenvironment, enhancing the local concentration of the tumor, minimizing the safety risks associated with systemic IL-2 exposure, and improving the safety of clinical treatment.

[0140] The ODD fragment of HIF-1α protein used in the present application has the following ODD amino acid sequence:

[0141] APAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQ (SEQ ID NO: 3).

[0142] IL-2 fusion protein

[0143] As used herein, the terms "fusion protein of the present application", "secreted fusion protein of the present application", "IL-2 fusion protein of the present application", and "secreted IL-2 fusion protein of the present application" are used interchangeably.

[0144] In a preferred embodiment, the fusion protein of the present application comprises: (1) an IL-2 element comprising IL-2 full length or an active fragment thereof; and (2) a CD25 element; and (3) an ODD element of HIF-1α protein.

[0145] In a preferred embodiment, the fusion protein of the present application has a structure as shown in formula (I):

[0146] Z1-L1-Z2-L2-Y (I)

[0147] wherein,

[0148] each of “-” is independently nothing or a connecting peptide;

[0149] Z1 is an IL-2 element;

[0150] L1 and L2 are each independently nothing or a linker;

[0151] Z2 is a CD25 element;

[0152] Y is an ODD element;

[0153] Preferably, the linker is a non-cleavable flexible linker, more preferably a GS linker peptide composed of glycine and serine. In a preferred embodiment, the linker amino acid sequence is as follows:

[0154] GGGGSGGGGSGGGGS (SEQ ID NO: 4).

[0155] In a preferred embodiment, the fusion protein comprises the following parts from N-terminus to C-terminus: an IL-2 element as shown in SEQ ID NO: 1, a CD25 element as shown in SEQ ID NO: 2, an ODD element as shown in SEQ ID NO: 3, and the three parts are connected by a linker as shown in SEQ ID NO: 4.

[0156] The fusion protein of the present application can fully exert the function of IL-2, enhance the activity after TIL infusion, and obtain longer persistence in vivo. At the same time, IL-2 is connected to soluble CD25 by a non-cleavable linker, which on the one hand, due to the increase in molecular weight, prolongs the half-life of IL-2 and increases the persistence of available IL-2 in circulation; on the other hand, the formed fusion protein cannot bind to CD25 on the surface of Treg cells, thereby further improving the tumor inhibitory immune microenvironment and enhancing the anti-tumor activity of TIL. In addition, the fusion expression design of the ODD domain of HIF-1α protein forms a double hypoxia sensing system molecular switch together with the hypoxia response element (HRE) in the promoter, which can ensure that IL-2 is only expressed in a hypoxic environment, thereby realizing the specific concentration expression of IL-2 in the tumor microenvironment, enhancing the local concentration of IL-2 in the tumor, minimizing the safety risks brought by systemic IL-2 exposure, and improving the safety of clinical treatment.

[0157] The sequence of an exemplary fusion protein of the present application is as follows:

[0158] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLGGGGSGGGGSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQGGGGSGGGGSGGGGSAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQ (SEQ ID NO: 5)

[0159] Hypoxia responsive element (HRE)

[0160] Hypoxia responsive element (HRE) exists in the promoter of hypoxia regulated target genes, which can sense the change of oxygen concentration in the environment and regulate the expression level of target genes. Under hypoxic conditions, HIF-1α and HIF-1β heterodimer complex act as transcription activators, enter the nucleus, bind to the DNA sequence of HRE in the promoter of target genes, and initiate the expression of target genes.

[0161] In a preferred embodiment of the present application, a promoter containing n hypoxia responsive elements (HRE) is constructed, i.e. a promoter containing n x HRE elements, wherein n is an integer selected from 3 to 9, preferably n is 9.

[0162] An exemplary HRE-containing promoter of the present application is an IL-12-mini promoter containing 9 x HRE elements, the nucleotide sequence of which is as follows:

[0163] TGTCACGTCCTGCACGACTCTAGTTGTTGTCACGTCCTGCACGACTCTAGTTGTCACGTCCTGCACGACGCTAGCACCCGGGAGGTACCGAGCTCTGTCACGTCCTGCACGACTCTAGTTGTCACGTCCTGCACGACTCTAGTTGTCACGTCCTGCACGACGCTAGCTACCGAGCTCTGTCACGTCCTGCACGACTCTAGTTGTCACGTCCTGCACGACTCTAGTTGTCACGTCCTGCACGACGCTAGCTCGAGATCCGGCCCCGCCCAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCGAGGTCCACCATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGC (SEQ ID NO: 9).

[0164] TIM-3

[0165] T cell immunoglobulin domain and mucin domain-3 (TIM-3) is an important immune checkpoint molecule and a key target in tumor immunotherapy. Similar to PD-1, it is also highly expressed on exhausted T cells and NK cells in chronic infection and cancer. High expression of TIM-3 on CD8+ T cells and NK cells is associated with the exhausted phenotype, while expression of TIM-3 on tumor-associated FoxP3+ regulatory T cells (Tregs) marks a subset of Tregs with stronger inhibitory function and recovery capacity. In the observation of PD-1 resistant patients, multiple emerging alternative immune checkpoints including TIM-3 are found to be compensatorily up-regulated, and the reactivity of anti-PD-1 therapy is restored when TIM-3 is combined with PD-1 in the treatment regimen

Br J Cancer. 2021; 125(7): 927-938

[0166] In the present application, the expression level of TIM-3 in TIL cells is greatly knocked down by introducing TIM-3 shRNA, thereby relieving the immune suppression induced by TIM-3.

[0167] In a preferred embodiment, the shRNA sequence having the TIM-3 silencing effect is as follows:

[0168] GCACCGCTGCTGCTGCTACTACTTACCTCGAGGTAAGTAGTAGCAGCAGCAGCTTTTTT (SEQ ID NO: 6).

[0169] Nucleic acid construct and expression vector of the present application

[0170] The present application provides a nucleic acid construct for preparing the engineered TIL cell of the present application. In an embodiment of the present application, the nucleic acid construct comprises a first expression cassette comprising the coding sequence of the aforementioned secretory IL-2 fusion protein; and further comprises a first promoter regulating the expression of the secretory IL-2 fusion protein in the first expression cassette, wherein the first promoter is a promoter comprising hypoxia response elements (HREs), preferably a promoter comprising n hypoxia response elements (HREs), i.e., a promoter comprising n x HRE elements, wherein n is an integer selected from 3-9, preferably n is 9. In addition, the nucleic acid construct further comprises a second expression cassette comprising the coding sequence of a shRNA targeting the endogenous TIM-3 transcript.

[0171] In a preferred embodiment of the present application, the first expression cassette and the second expression cassette are integrated into a lentiviral vector in series to obtain an expression vector, and the expression vector is introduced into a TIL cell to obtain the engineered TIL cell of the present application.

[0172] Specifically, the expression vector of the present application contains a nucleic acid construct as shown in formula (IV):

[0173] P2-R-P1-SP-Z1’-L1’-Z2’-L2’-Y’ (IV)

[0174] In the formula,

[0175] Each of “-” is independently a connecting bond;

[0176] P2 is a second promoter, preferably the second promoter is a U6 promoter, and the nucleotide sequence of the U6 promoter is as shown in SEQ ID NO: 7;

[0177] R is the coding sequence of a shRNA targeting the endogenous TIM-3 transcript, preferably the sequence of the shRNA is as shown in SEQ ID NO: 6;

[0178] P1 is a first promoter, preferably the first promoter is an IL-12-mini promoter comprising 9 x HRE elements, and the nucleotide sequence of the IL-12-mini promoter is as shown in SEQ ID NO: 9;

[0179] SP is a coding sequence of a signal peptide, preferably the coding sequence of the signal peptide is set forth in SEQ ID NO: 10;

[0180] Z1’ is a coding sequence of an IL-2 element, preferably the coding sequence of the IL-2 element is set forth in SEQ ID NO: 11;

[0181] L1’ and L2’ are each independently a coding sequence of nothing or a linker, preferably the coding sequence of the linker is set forth in SEQ ID NO: 12;

[0182] Z2’ is a coding sequence of a CD25 element, preferably the coding sequence of the CD25 element is set forth in SEQ ID NO: 13;

[0183] Y’ is a coding sequence of an oxygen-dependent degradation domain (ODD) element, preferably the coding sequence of the ODD element is set forth in SEQ ID NO: 14.

[0184] Engineered TIL cells

[0185] As used herein, the terms “engineered TIL cells of the invention” and “TIL cells of the invention” are used interchangeably and refer to TIL cells containing the expression vector of the invention, capable of expressing the secreted IL-2 fusion protein of the invention under hypoxic conditions.

[0186] The introduction of TIM-3 shRNA in the engineered TIL cells of the invention effectively knocks down the expression level of TIM-3 in the TIL cells, reduces the TIL cell exhaustion mediated by the TIM-3 / Galectin-9 pathway; and the IL-2 fusion protein in the cells is designed with a dual hypoxia-inducible safety switch, which controls the expression of the IL-2 fusion protein only under hypoxic conditions, on the one hand, it realizes the specific concentration expression of IL-2 in the tumor microenvironment, enhances the local concentration in the tumor, and effectively promotes the growth of TILs in the tumor tissue; on the other hand, it maximizes the safety risks brought by systemic IL-2 exposure, and improves the safety of clinical treatment.

[0187] Cell preparation

[0188] The cell preparation of the invention comprises the engineered TIL cells of the invention and one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants. The cell preparation can be suitable for use in the treatment of the therapeutic indications described herein.

[0189] The cell preparation can be a liquid solution, suspension, emulsion, or sustained release formulation, and can be formulated with a pharmaceutically acceptable carrier. The formulation can be formulated into a suppository using traditional binders 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 ingredients and that is not toxic to the host or subject.

[0190] The engineered TIL cells can be delivered 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, noisomes, nanosomes, etc.), microparticles or microcapsules, or colloidal formulations comprising pharmaceutically acceptable polymers.

[0191] The cell preparation can be formulated into a sterile injectable aqueous or oleaginous suspension in accordance with known methods in the art and using suitable dispersing or wetting agents and / or suspending agents. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.

[0192] In the present application, the term "adjuvant" generally refers to any substance that assists or modulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen.

[0193] In the present application, the term "subject" can be a mammal in need of treatment, such as a human or a veterinary patient (e.g., a rodent, such as a mouse or rat, a cat, a dog, a cow, a horse, a sheep, a goat, or other livestock). In some embodiments, a "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like. The subject can 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, methods for diagnosis of a disease characterized by cell proliferation and clinical partitioning of such diagnosis are known to those of skill in the art.

[0194] The cell preparation of the present application can be used to treat a tumor. In the present application, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of tumors such as solid tumors include, but are not limited to, lung cancer, cervical cancer, melanoma, colorectal cancer, head and neck cancer, liver cancer, ovarian cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, prostate cancer, esophageal cancer, and breast cancer.

[0195] The primary advantages of the present application include:

[0196] (1) The IL-2 fusion protein expressed by the nucleic acid construct of the present application after being introduced into TILs can selectively bind to the medium affinity IL-2R receptor, thereby selectively activating the expansion of CD8+ T cells and NK cells and inhibiting the activity of Treg cells, thereby enhancing the anti-tumor ability of TIL cells.

[0197] (2) The nucleic acid construct of the present application adopts a double hypoxia-sensitive safety switch design to control the expression of the fusion protein only in a hypoxic environment. On the one hand, it realizes the specific concentration of IL-2 expression in the tumor microenvironment, enhances the local concentration of the tumor, and effectively promotes the growth of TILs in the tumor tissue. On the other hand, it maximizes the safety risks brought by systemic IL-2 exposure and improves the safety of clinical treatment.

[0198] (3) The nucleic acid construct of the present application introduces TIM-3 shRNA, which can effectively knock down the expression level of TIM-3 in TIL cells and reduce the TIL cell exhaustion mediated by the TIM-3 / Galectin-9 pathway.

[0199] The present application will be further illustrated in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples are not specified, and the percentages and fractions are quantitative percentages, unless otherwise specified, according to the conventional conditions, such as the conditions 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.

[0200] Example 1: Lentivirus containing the target gene infects TIL cells

[0201] (1) Preparation of lentivirus vector

[0202] The structure of the nucleic acid construct expressed on each of the 1#, 2#, 3#, 4# and 5# TIL cells is shown in Figure 1 The nucleotide sequences of the elements in the figure are as follows:

[0203] U6 promoter: GAGGGCCTATTTCCCATGATTCCTTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC (SEQ ID NO: 7)

[0204] SFFV promoter: GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGG (SEQ ID NO: 8)

[0205] Signal peptide (SP): ATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCATCATCT (SEQ ID NO: 10)

[0206] IL-2: ATGTATCGCATGCAGCTGCTGAGCTGCATTGCGCTGAGCCTGGCGCTGGTGACCAACAGCGCGCCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAGCTGGAACATCTGCTGCTGGATCTGCAGATGATTCTGAACGGCATTAACAACTATAAAAACCCGAAACTGACCCGCATGCTGACCTTTAAATTTTATATGCCGAAAAAAGCGACCGAACTGAAACATCTGCAGTGCCTGGAAGAAGAACTGAAACCGCTGGAAGAAGTGCTGAACCTGGCGCAGAGCAAAAACTTTCATCTGCGCCCGCGCGATCTGATTAGCAACATTAACGTGATTGTGCTGGAACTGAAAGGCAGCGAAACCACCTTTATGTGCGAATATGCGGATGAAACCGCGACCATTGTGGAATTTCTGAACCGCTGGATTACCTTTTGCCAGAGCATTATTAGCACCCTG (SEQ ID NO: 11)

[0207] Linker 1 and Linker 2: GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC (SEQ ID NO: 12)

[0208] CD25: GAGCTCTGTGACGATGACCCGCCAGAGATCCCACACGCCACATTCAAAGCCATGGCCTACAAGGAAGGAACCATGTTGAACTGTGAATGCAAGAGAGGTTTCCGCAGAATAAAAAGCGGGTCACTCTATATGCTCTGTACAGGAAACTCTAGCCACTCGTCCTGGGACAACCAATGTCAATGCACAAGCTCTGCCACTCGGAACACAACGAAACAAGTGACACCTCAACCTGAAGAACAGAAAGAAAGGAAAACCACAGAAATGCAAAGTCCAATGCAGCCAGTGGACCAAGCGAGCCTTCCAGGTGAAGAGAAGCCTCAGGCAAGCCCCGAAGGCCGTCCTGAGAGTGAGACTTCCTGCCTCGTCACAACAACAGATTTTCAAATACAGACAGAAATGGCTGCAACCATGGAGACGTCCATATTTACAACAGAGTACCAG (SEQ ID NO: 13)

[0209] ODD: GCGCCGGCGGCGGGCGATACCATTATTAGCCTGGATTTTGGCAGCAACGATACCGAAACCGATGATCAGCAGCTGGAAGAAGTGCCGCTGTATAACGATGTGATGCTGCCGAGCCCGAACGAAAAACTGCAGAACATTAACCTGGCGATGAGCCCGCTGCCGACCGCGGAAACCCCGAAACCGCTGCGCAGCAGCGCGGATCCGGCGCTGAACCAGGAAGTGGCGCTGAAACTGGAACCGAACCCGGAAAGCCTGGAACTGAGCTTTACCATGCCGCAGATTCAGGATCAGACCCCGAGCCCGAGCGATGGCAGCACCCGCCAGAGCAGCCCGGAACCGAACAGCCCGAGCGAATATTGCTTTTATGTGGATAGCGATATGGTGAACGAATTTAAACTGGAACTGGTGGAAAAACTGTTTGCGGAAGATACCGAAGCGAAAAACCCGTTTAGCACCCAGGATACCGATCTGGATCTGGAAATGCTGGCGCCGTATATTCCGATGGATGATGATTTTCAGCTGCGCAGCTTTGATCAGCTGAGCCCGCTGGAAAGCAGCAGCGCGAGCCCGGAAAGCGCGAGCCCGCAGAGCACCGTGACCGTGTTTCAGTAA (SEQ ID NO: 14)

[0210] The gene sequence of each nucleic acid construct was directly synthesized by GeneScript and cloned into a lentiviral expression vector pCDH-EF1a-MCS, replacing the sequence from the EF1a promoter to the MCS in the vector with the target gene sequence. Each vector plasmid was then mixed with lentiviral packaging plasmids pMDLg-pRRE, pRSV-Rev, and pMD2.G, and transfected into 293T cells using Lipofectamine 3000 reagent according to the instructions. After 4 hours of transfection, the cells were removed, the supernatant was discarded, and complete medium was added for continued culture for 48 hours. The transfected viral supernatant was then harvested, filtered and concentrated, and aliquoted for storage at -80°C.

[0211] (2) Preparation of lung cancer TIL cells

[0212] Fresh lung cancer tumor tissues were removed after surgery, and necrotic tissues and connective tissues were removed under sterile conditions and washed twice with normal saline. The tissues were cut into about 1-27 mm 3 tumor fragments, and an appropriate amount of tumor dissociation kit (Tumor Dissociation Kit, purchased from Miltenyi, Cat. No. 130-095-929) was added. The tumor fragments were lysed at 37°C using an automatic tissue processor (gentle MACS Octo Dissociator with Heaters, Cat. No. 130-096-427, purchased from Miltenyi) for 1 min of mechanical lysis every half hour, followed by 29 min of standing, for a total of 2 cycles. After the tumor tissue fragments were lysed by the automatic tissue processor, they were filtered through a 70 μm filter, and the lower single cell suspension was collected. After the collected single cell suspension was cultured in the medium containing ketorolac and IL-2 for about 2 weeks, the harvested suspension cells were TIL seed cells.

[0213] (3) Lentivirus infection of TIL cells

[0214] TIL seed cells (3 x 10 6 ) were resuspended in 6 ml of REP complete medium, corresponding lentivirus (MOI = 60) and 5 μg / ml of polybrene were added, mixed and transferred to a 6-well plate, and cultured in a 37°C, 5% CO2 incubator. After 24 hours of infection, the cells were collected and centrifuged to remove the virus solution. The TIL cells after lentivirus transfection were designated as 1#TIL, 2#TIL, 3#TIL, 4#TIL and 5#TIL, and the TIL cells without transduction of lentivirus were designated as Ctrl TIL.

[0215] Example 2: Detection of enhanced TIL cells

[0216] (1) Detection of IL-2 secretion in TIL cells in each group

[0217] 1 x 10 6 TIL, 3#TIL, 4#TIL, 5#TIL and control TIL (Ctrl TIL) cells prepared in Example 1 were transferred to a new 24-well plate and cultured in medium without additional IL-2 under normal conditions (37°C, 5% CO2 incubator, oxygen concentration 21%) and hypoxic conditions (culture in a hypoxic chamber, oxygen concentration 1%) for 24 hours. The supernatant was collected and subjected to ELISA to determine the secretion of IL-2 in each group of TIL cells. The kit used was a human IL-2 pre-prepared ELISA kit (Yeasen, cat. 97029ES96), and the specific detection steps were performed according to the corresponding ELISA kit instructions.

[0218] IL-2 secretion test results are as follows Figure 2 As shown: Under normal circumstances (see...) Figure 2 In Figure A), the unmodified control TIL (Ctrl TIL) cells secreted very low levels of IL-2. TIL #5, using a constitutive promoter, showed the highest IL-2 secretion. TIL #3, employing a 9×HRE-IL-2 mini inducible promoter and an ODD fusion expression dual hypoxia-sensing switch design, secreted IL-2 levels comparable to the control TIL (Ctrl TIL). Notably, TIL #4, using only a 9×HRE-TK inducible promoter single hypoxia-sensing system design, resulted in significantly higher IL-2 secretion levels than TIL #3. These results suggest that a dual hypoxia-sensing system design is more robust than a single hypoxia-sensing switch design, allowing for stricter control of target protein synthesis and secretion under hypoxic conditions. Under hypoxic conditions (see...), Figure 2 In the study (B), TILs 1, 3, 4, and 5 all secreted high concentrations of IL-2, significantly higher than the control TIL (Ctrl TIL). However, TIL 5 was significantly lower than TILs 1, 3, and 4. The results suggest that the IL-2 fusion protein has a longer half-life than IL-2 alone, resulting in a higher concentration of IL-2 accumulated in the supernatant.

[0219] (2) Flow cytometry was used to detect the expression level of TIM-3 in TIL cells of each group.

[0220] Take 1×10TIL each of the 1#TIL, 2#TIL, 3#TIL and control TIL (Ctrl TIL) cells prepared in Example 1. 6 The cells were transferred to new 24-well plates and cultured for 24 hours under hypoxic conditions in medium without additional IL-2. Cells were then collected. After washing twice with PBS, TILs 1#, 2#, 3#, and the control TIL (Ctrl TIL) were labeled with APC anti-human CD336 (TIM-3) antibody (Biolegend, cat. 345011) and their corresponding isotype controls, respectively. Cells were incubated at room temperature in the dark for 15 minutes, followed by washing twice with PBS. The supernatant was discarded, and the cells were resuspended in 200 μl of PBS for analysis.

[0221] The results are as follows Figure 3 As shown, compared with the control TIL (Ctrl TIL), the expression of TIM-3 in 1#TIL and 2#TIL cells was significantly reduced, while the expression of TIM-3 in 3#TIL and control TIL (Ctrl TIL) cells was not significantly different from that in the control group.

[0222] The results show that TIM-3 shRNA can silence the expression of TIM-3 gene in TIL cells infected by lentivirus.

[0223] (4) Flow cytometry detection of TIL cell subtypes in each group

[0224] The harvested 1# TIL seed cells after lentivirus transfection in Example 1 were expanded and cultured under hypoxic conditions. The culture system contained 40 ng / ml OKT3 antibody, and irradiated allogeneic PBMC (PBMC cell number was 200 times that of TIL cells) were used as feeder cells to co-culture with the lentivirus-transfected TIL seed cells for 14 days. No additional IL-2 was added during the culture process. Meanwhile, 3000 IU / ml IL-2 was added to the Ctrl TIL culture system as a positive control group, and the other culture conditions were consistent with those of the other groups. After the culture ended, flow cytometry was used to detect the proportions of Treg, CD4+, and CD8+ cells in the cells.

[0225] The flow cytometry results are shown in Figure 4 The proportion of CD4+ T cells in the Ctrl TIL cells was significantly higher than that in the 1# TIL group, and the proportion of FOXP3+ CD25+ cells (Treg cell phenotype characteristic CD4+ FOXP3+ CD25+) in the CD4+ T cells was significantly higher than that in the 1# TIL group.

[0226] The results show that the IL-2 fusion protein secreted by the genetically modified 1# TIL can more effectively and selectively promote the expansion of CD8+ T lymphocytes and inhibit the expansion of Treg cells compared to conventional IL-2.

[0227] (5) Expansion and viability of TIL cells under hypoxic conditions

[0228] The harvested 1# TIL seed cells after lentivirus transfection in Example 1 and Ctrl TIL were expanded and cultured under hypoxic conditions. The culture system contained 40 ng / ml OKT3 antibody, and irradiated allogeneic PBMC (PBMC cell number was 200 times that of TIL cells) were used as feeder cells to co-culture with the lentivirus-transfected TIL seed cells for 14 days. No additional IL-2 was added during the culture process. After the culture ended, the proliferation fold and viability of TIL cells in each group were detected. After the culture ended, the cell proliferation fold and viability results are shown in Tables 1 and 2, which show the results of three parallel experiments.

[0229] Table 1 TIL cell proliferation fold

[0230]

[0231] Table 2 TIL cell viability

[0232]

[0233] The 1#TIL proliferation fold and cell viability were significantly higher than the Ctrl TIL without additional IL-2 under hypoxia. The results suggest that the genetically modified 1#TIL can secrete sufficient IL-2 to promote TIL cell proliferation and maintain TIL cell viability under hypoxic conditions.

[0234] Example 3: Tumor killing ability of TIL in each group

[0235] To verify the difference in function of TIL cells overexpressing IL-2 fusion protein and silencing TIM-3 gene expression on anti-tumor, the 1#TIL, 2#TIL, 3#TIL and Ctrl TIL cells prepared in Example 1 were cultured under normal conditions, and the culture system contained 3000 IU / ml IL-2, 40 ng / ml OKT3 antibody, and irradiated allogeneic PBMC (PBMC cell number was 200 times that of TIL cells) for expansion culture for 14 days. After the culture ended, the corresponding TIL cells harvested were mixed with corresponding autologous tumor cells at an effector target ratio of 30:1, 10:1, and 5:1, and cultured in a 96-well plate with a total volume of 200 μl per well. The spontaneous release well (blank control) and maximum release well were set in the 96-well plate. The target cell natural release well added 100 μl of target cells and culture medium, and the target cell maximum release well added 100 μl of target cells and 2.5% Triton X-100. After 24 h of culture under hypoxic conditions, the 96-well plate was centrifuged at 250 g for 5 min, 100 μl of supernatant was taken from each well and placed in a flat-bottom 96-well plate, 100 μl of LDH substrate solution was added, the reaction was allowed to proceed for 5 min, 30 μl of HCl (1 mol / L) was added to each well, and the absorbance was measured at 490 nm on a microplate reader.

[0236] The cytotoxicity / killing rate was calculated according to the method described in the kit instructions = (absorbance of the sample to be tested - absorbance of the blank control) / (absorbance of the maximum release well - absorbance of the blank control) x 100%.

[0237] The results are shown in Figure 5 As shown in Table 3, under hypoxic conditions, the killing rate of the unmodified Ctrl TIL on tumor cells was limited, while the 1#TIL, 2#TIL, and 3#TIL cells showed significantly higher killing toxicity on target cells than the Ctrl TIL after co-incubation with autologous tumor cells, and the killing effect of the 1#TIL was better than that of the 2#TIL and 3#TIL cells. In addition, the killing toxicity of the 1#TIL on target cells showed a synergistic anti-tumor effect of overexpressing IL-2 fusion protein and silencing TIM-3 gene expression.

[0238] Table 3 Tumor cell killing rates of TILs from each group

[0239]

[0240] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits disclosed herein.

Claims

1. An engineered TIL cell, characterized in that, The engineered TIL cells have the following characteristics: (a) The TIL cells described herein express exogenous secretory IL-2 fusion protein in a hypoxic environment under the regulation of a promoter containing a hypoxia-responsive element (HRE); and (b) The expression of endogenous TIM-3 in the TIL cells was silenced; The engineered TIL cells comprise a nucleic acid construct having the structure shown in Formula (IV) from the 5' end to the 3' end: P2-R-P1-SP-Z1'-L1'-Z2'-L2'-Y' (IV) In this context, each "-" represents a separate connector. P2 is the second promoter; R is the coding sequence of shRNA that targets endogenous TIM-3 transcripts; P1 is the first promoter, which is a promoter containing n hypoxia response elements (HREs), where n is an integer selected from 3 to 12; SP is the coding sequence for the signal peptide; Z1' is the coding sequence of the IL-2 element, and the amino acid sequence of the IL-2 element is shown in SEQ ID NO: 1; L1' and L2' are each independently encoded sequences without any connectors; Z2' is the coding sequence of the CD25 element, and the amino acid sequence of the CD25 element is shown in SEQ ID NO: 2; Y' is the coding sequence of an oxygen-dependent degradation domain (ODD) element, the amino acid sequence of which is shown in SEQ ID NO:

3.

2. The engineered TIL cells as described in claim 1, characterized in that, The nucleotide sequence of the shRNA is shown in SEQ ID NO:

6.

3. The engineered TIL cells as described in claim 1, characterized in that, The encoding sequence of the IL-2 element is shown in SEQ ID NO: 11, the encoding sequence of the CD25 element is shown in SEQ ID NO: 13, and / or the encoding sequence of the ODD element is shown in SEQ ID NO:

14.

4. The engineered TIL cells as described in claim 3, characterized in that, The amino acid sequence of the secreted IL-2 fusion protein is shown in SEQ ID NO:

5.

5. The engineered TIL cells according to any one of claims 1-4, characterized in that, The promoter containing the hypoxia response element (HRE) is an IL-12-mini promoter containing 9×HRE elements, and its nucleotide sequence is shown in SEQ ID NO:

9.

6. An expression vector comprising a nucleic acid construct as shown in formula (IV): P2-R-P1-SP-Z1'-L1'-Z2'-L2'-Y' (IV) In the formula, Each "-" is a separate connector key; P2 is the second promoter; R is the coding sequence of shRNA that targets endogenous TIM-3 transcripts; P1 is the first promoter, which is a promoter containing n hypoxia response elements (HREs), where n is an integer selected from 3 to 12; SP is the coding sequence for the signal peptide; Z1' is the coding sequence of the IL-2 element, and the amino acid sequence of the IL-2 element is shown in SEQ ID NO: 1; L1' and L2' are each independently encoded sequences without any connectors; Z2' is the coding sequence of the CD25 element, and the amino acid sequence of the CD25 element is shown in SEQ ID NO: 2; Y' is the coding sequence of an oxygen-dependent degradation domain (ODD) element, the amino acid sequence of which is shown in SEQ ID NO:

3.

7. The expression vector as described in claim 6, characterized in that, The nucleotide sequence of the shRNA is shown in SEQ ID NO:

6.

8. The expression vector as described in claim 6, characterized in that, The encoding sequence of the IL-2 element is shown in SEQ ID NO: 11, the encoding sequence of the CD25 element is shown in SEQ ID NO: 13, and / or the encoding sequence of the ODD element is shown in SEQ ID NO:

14.

9. A method for preparing engineered TIL cells as described in claim 1, characterized in that, Includes the following steps: (1) Provide TIL cells to be modified; and (2) The TIL cells are modified such that the expression of endogenous TIM-3 in the TIL cells is silenced and the TIL cells contain the gene encoding the secretory IL-2 fusion protein, thereby obtaining the engineered TIL cells.

10. A cell preparation, characterized in that, The cell preparation contains engineered TIL cells as described in any one of claims 1-5, and a pharmaceutically acceptable carrier, diluent, or excipient.

11. Use of an engineered TIL cell as described in any one of claims 1-5 in the preparation of a medicament or formulation for the prevention and / or treatment of lung cancer.

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