Engineered til cell having improved tumor recognition and killing capability and use thereof

Abstract

Provided are an engineered TIL cell having an improved tumor recognition and killing capability and a use thereof, relating to the technical field of genetic engineering and cell therapy. The surface of the engineered TIL cell comprises an exogenous membrane-bound DAP10-CD3ζ fusion protein, and the endoplasmic reticulum or Golgi apparatus of the engineered TIL cell comprises a retained fusion protein. Specifically, provided is the membrane-bound DAP10-CD3ζ fusion protein, so that the TIL cell can be activated by an activated fusion protein when recognizing a tumor cell by means of the NKG2D-NKG2DL pathway, so as to effectively kill the tumor cell. Further provided is the retained fusion protein, so that endogenous NKG2DL that is expressed when the TIL cell is activated can bind to the retained fusion protein so as to be retained in the cell, thereby avoiding mutual recognition and killing between the TIL cells and improving the viability of the TIL cells.

Description

Engineered TIL cells with enhanced tumor recognition and killing capabilities and their applications Technical Field

[0001] The present invention relates to the technical field of genetic engineering and cell therapy, and in particular to engineered TIL cells with improved tumor recognition and killing capabilities and applications thereof. Background Art

[0002] Tumor-infiltrating lymphocyte (TIL) cell therapy refers to a therapy that separates tumor-infiltrating lymphocytes from tumor tissue, cultures them in vitro, expands them in large quantities, and then infuses them back into the patient's body. TIL is composed of T cells with multiple TCR clones, which can recognize multiple tumor-specific neoantigens and tumor-associated antigens, making it more effective in dealing with tumor heterogeneity. TIL usually contains a certain amount of effector memory T cells, which express chemokine receptors after being stimulated by tumor antigens in vivo, making it easier for them to localize to tumor tissue after infusion. In addition, TIL is derived from the patient himself and has lower toxicity. TIL therapy has shown great potential in the field of solid tumors.

[0003] TIL cells derived from tumor tissue are rich in tumor-specific T cells. However, after tumor-specific activation, these tumor-specific T cells are often in a terminally differentiated and exhausted state, resulting in a weakened ability of TIL cells to kill tumor cells. In addition, the majority of cultured TIL cells are non-tumor-specific bystander cells. How to transform bystander cells into effector cells with the ability to recognize and kill tumor cells is an urgent problem to be solved. In addition, tumor cells often reduce the expression of MHC-I molecules or mutate the TAP gene, resulting in loss of antigen presentation function, thereby escaping the killing of immune cells.

[0004] NKG2D molecules on the surface of TIL cells can bind to NKG2DL molecules on the surface of tumor cells, allowing TIL cells to recognize tumor cells. However, when activated, TIL cells briefly express NKG2DL, which causes mutual killing between TIL cells and a significant decrease in TIL cell activity.

[0005] Therefore, there is an urgent need in this field to develop engineered TIL cells that have the ability to recognize and kill tumor cells, reduce immune escape, and reduce TIL cell cannibalism. Summary of the Invention

[0006] The purpose of the present invention is to provide engineered TIL cells and their applications that have the function of recognizing and killing tumor cells, reducing immune escape, and reducing TIL cell cannibalism.

[0007] In a first aspect of the present invention, an engineered tumor-infiltrating lymphocyte (TIL cell) is provided, wherein the engineered TIL cell expresses an exogenous fusion protein selected from the group consisting of an activated fusion protein, a retained fusion protein, or a combination thereof;

[0008] Wherein, the activated fusion protein is a membrane-bound DAP10-CD3ζ fusion protein;

[0009] The retention-type fusion protein includes: an NKG2D extracellular region and a KDEL sequence.

[0010] In another preferred embodiment, the engineered TIL cells express an activated fusion protein and a retained fusion protein.

[0011] In another preferred embodiment, the activated fusion protein comprises: (1) an extracellular domain, wherein the extracellular domain comprises a DAP10 element or an active fragment thereof;

[0012] (2) transmembrane domain; and

[0013] (3) an intracellular signaling domain comprising the DAP10 intracellular domain and CD3ζ;

[0014] Wherein, the extracellular domain and the intracellular signaling domain are connected in series through the transmembrane domain.

[0015] In another preferred embodiment, the engineered TIL cells have the activated fusion protein on their cell membrane; and / or the engineered TIL cells have the retained fusion protein in their endoplasmic reticulum or Golgi apparatus.

[0016] In another preferred embodiment, the engineered TIL cells contain a polynucleotide encoding the activated fusion protein and / or the retained fusion protein.

[0017] In another preferred embodiment, the engineered TIL cells contain a nucleic acid construct, which comprises a first expression cassette, which comprises a polynucleotide encoding the activation-type fusion protein and / or a polynucleotide encoding the retention-type fusion protein.

[0018] In another preferred embodiment, the engineered TIL cells contain a vector comprising: a polynucleotide encoding the activated fusion protein and / or a polynucleotide encoding the retained fusion protein.

[0019] In another preferred embodiment, the engineered TIL cells further express a membrane-bound IL-15 fusion protein comprising the following elements:

[0020] (i) interleukin 15 or a functionally active fragment thereof;

[0021] (ii) a transmembrane domain; and

[0022] (iii) intracellular domain;

[0023] Wherein, the intracellular domain is the CD86 intracellular domain.

[0024] In another preferred embodiment, the CD86 intracellular domain includes the full-length intracellular domain, or an active fragment thereof.

[0025] In another preferred embodiment, the IL-15 comprises a full-length, mature form of IL-15, or an active fragment thereof.

[0026] In another preferred embodiment, the IL-15 includes wild type and mutant type.

[0027] In another preferred embodiment, the amino acid sequence of IL-15 is shown in SEQ ID NO: 6.

[0028] In another preferred embodiment, the fusion polypeptide further comprises a hinge region.

[0029] In another preferred embodiment, the fusion polypeptide may further optionally include a signal peptide and / or a linker.

[0030] 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).

[0031] In another preferred embodiment, the linker includes a flexible linker or a rigid linker.

[0032] In another preferred embodiment, the amino acid sequence of the linker is as shown in SEQ ID NO: 7.

[0033] In another preferred embodiment, the structural formula of the fusion polypeptide is as shown in formula (I): X-IL15-LH-TM-Cyto (I)

[0034] in,

[0035] “-” each independently represents no peptide or a connecting peptide;

[0036] X is none or signal peptide;

[0037] IL15 is the interleukin 15 element;

[0038] L is none or a linker;

[0039] H is the hinge region;

[0040] TM is the transmembrane domain;

[0041] Cyto is an intracellular domain, wherein the intracellular domain is the CD86 intracellular domain.

[0042] In another preferred embodiment, the transmembrane domain is the CD86 transmembrane domain.

[0043] In another preferred example, the amino acid sequences of the CD86 transmembrane domain and intracellular domain are as shown in SEQ ID NO:9.

[0044] 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:7.

[0045] In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 5.

[0046] In another preferred embodiment, the engineered TIL cells have one or more properties selected from the following group:

[0047] (1) Enhanced ability to recognize and kill tumor cells;

[0048] (2) Reduce the ability of activated TIL cells to recognize and kill each other;

[0049] (3) expanded TIL cell recognition of target antigen spectrum;

[0050] (4) Effectively prevent tumor immune escape and recurrence.

[0051] In a second aspect of the present invention, an activated fusion protein is provided, wherein the activated fusion protein comprises:

[0052] (1) an extracellular domain comprising a DAP10 element or an active fragment thereof;

[0053] (2) transmembrane domain; and

[0054] (3) an intracellular signaling domain comprising the DAP10 intracellular domain and CD3ζ;

[0055] Wherein, the extracellular domain and the intracellular signaling domain are connected in series through the transmembrane domain.

[0056] In another preferred embodiment, in the intracellular signaling domain, the DAP10 intracellular domain and CD3ζ are directly connected or connected via a connecting peptide (preferably, the length of the connecting peptide is 1-10 aa, more preferably 1-6 aa, and more preferably 1-3 aa).

[0057] In another preferred embodiment, the intracellular signaling domain further includes a costimulatory domain.

[0058] In another preferred embodiment, the intracellular signaling domain further includes one, two or more costimulatory domains.

[0059] In another preferred embodiment, the costimulatory domain is a costimulatory domain from a protein selected from the group consisting of CD28, CD27, 4-1BB, CD40, OX40, or a combination thereof.

[0060] In another preferred embodiment, the costimulatory domain is located between the DAP10 intracellular domain and CD3ζ, and / or is located at the C-terminus of CD3ζ.

[0061] In another preferred embodiment, when the intracellular signaling domain contains an additional costimulatory domain, the DAP10 intracellular domain and the costimulatory domain, and / or CD3ζ and the costimulatory domain, can be directly connected or connected through a connecting peptide; preferably, the length of the connecting peptide is 1-10 aa, more preferably 1-6 aa, and most preferably 1-3 aa.

[0062] In another preferred embodiment, the fusion protein further contains a signal peptide at the N-terminus.

[0063] In another preferred embodiment, the signal peptide is selected from the following group: DAP10 signal peptide, CD8a signal peptide, CD28 signal peptide.

[0064] In another preferred embodiment, the amino acid sequence of the signal peptide is as shown in positions 1-18 of SEQ ID NO: 1.

[0065] In another preferred embodiment, the amino acid sequence of the extracellular domain is as shown in positions 19-48 of SEQ ID NO: 1.

[0066] In another preferred embodiment, the transmembrane domain is a transmembrane domain from DAP10, CD8a, or CD28.

[0067] In another preferred embodiment, the amino acid sequence of the transmembrane domain is as shown in positions 49-69 of SEQ ID NO: 1.

[0068] In another preferred embodiment, the amino acid sequence of the DAP10 intracellular domain in the intracellular signaling domain is as shown in positions 70-92 of SEQ ID NO: 1.

[0069] In another preferred embodiment, the amino acid sequence of CD3ζ in the intracellular signaling domain is as shown in positions 93-206 of SEQ ID NO: 1.

[0070] In another preferred embodiment, the amino acid sequence of the intracellular signaling domain is as shown in positions 70-206 of SEQ ID NO: 1.

[0071] In another preferred embodiment, the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO: 1.

[0072] In another preferred embodiment, the fusion polypeptide has one or more properties selected from the following group:

[0073] (a) The fusion protein has the function of binding to NKG2D;

[0074] (b) activated T cells (TIL cells);

[0075] (c) Activation of NK cells.

[0076] In another preferred embodiment, the activation is self-activation (ie, the immune cells expressing the membrane-bound DAP10-CD3ζ fusion protein are activated).

[0077] In another preferred embodiment, the activated immune cells (T cells, NK cells) can effectively kill target cells through the interaction of NKG2D-NKG2DLs.

[0078] In a third aspect of the present invention, a retention-type fusion protein is provided. The retention-type fusion protein comprises: an NKG2D extracellular region and a KDEL sequence.

[0079] In another preferred embodiment, the KDEL sequence of the retention-type fusion protein is located at the C-terminus of the NKG2D extracellular region.

[0080] In another preferred embodiment, in the retention-type fusion protein, the NKG2D extracellular region is as shown in positions 22-156 of SEQ ID NO: 2.

[0081] In another preferred embodiment, the NKG2D extracellular region and the KDEL sequence are directly connected or connected through a connecting peptide; preferably, the length of the connecting peptide is 1-15 aa, more preferably 1-10 aa, and most preferably 1-5 aa.

[0082] In another preferred embodiment, the KDEL sequence is located at positions 157-160 in SEQ ID NO: 2.

[0083] In another preferred embodiment, the amino acid sequence of the retention-type fusion protein is shown in SEQ ID NO: 2.

[0084] In another preferred embodiment, the retention-type fusion protein further comprises a signal peptide; preferably a CD8a signal peptide; more preferably, the signal peptide is as shown in SEQ ID NO: 3.

[0085] In another preferred embodiment, the retention-type fusion protein can bind to endogenous NKG2DL, causing the endogenous NKG2DL to be retained in the cell.

[0086] In the fourth aspect of the present invention, a polynucleotide is provided, which encodes the activation-type fusion protein described in the second aspect of the present invention and / or the retention-type fusion protein described in the third aspect of the present invention.

[0087] In another preferred embodiment, the polynucleotide encodes the activated fusion protein described in the second aspect of the present invention.

[0088] In another preferred embodiment, the polynucleotide encodes the retention-type fusion protein described in the third aspect of the present invention.

[0089] In another preferred embodiment, the polynucleotide encodes the activation-type fusion protein described in the second aspect of the present invention and the retention-type fusion protein described in the third aspect of the present invention.

[0090] In another preferred embodiment, the activation-type fusion protein and the retention-type fusion protein are connected via a cleavable connecting peptide.

[0091] In another preferred embodiment, the cleavable connecting peptide is a self-cleaving peptide; preferably, the self-cleaving peptide is selected from the following group: T2A peptide, P2A peptide, or a combination thereof.

[0092] In the fifth aspect of the present invention, a nucleic acid construct is provided, wherein the nucleic acid construct comprises a first expression cassette, wherein the first expression cassette comprises the polynucleotide according to the fourth aspect of the present invention.

[0093] In another preferred embodiment, the first expression cassette further contains a promoter.

[0094] In another preferred embodiment, the promoter is operably linked to the polynucleotide.

[0095] In another preferred embodiment, the promoter is selected from the following group: a constitutive promoter, an inducible promoter or a combination thereof.

[0096] In another preferred embodiment, the promoter includes a hypoxia-responsive promoter.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] In another preferred embodiment, the hypoxia-responsive promoter is a 3×HRE TK-mini promoter.

[0101] 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.

[0102] In another preferred embodiment, the molecular switch element is hEGFRt.

[0103] In another preferred embodiment, the molecular switch element is connected to the first expression cassette via a cleavable connecting peptide sequence.

[0104] In another preferred embodiment, the cleavable connecting peptide is a self-cleaving 2A peptide, preferably a T2A peptide.

[0105] 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.

[0106] In another preferred embodiment, the second promoter is the SFFV promoter.

[0107] In the sixth aspect of the present invention, a vector is provided, wherein the vector comprises the polynucleotide according to the fourth aspect of the present invention.

[0108] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0109] In another preferred embodiment, the viral vector includes: a lentiviral vector, an adenoviral vector, and a yellow fever virus vector.

[0110] In another preferred embodiment, the vector is a plasmid.

[0111] In the seventh aspect of the present invention, a composition is provided, which comprises the engineered TIL cells described in the first aspect of the present invention, the activated fusion protein described in the second aspect of the present invention, the retained fusion protein described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the nucleic acid construct described in the fifth aspect of the present invention, and / or the vector described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0112] In another preferred embodiment, the composition comprises the engineered TIL cells described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0113] In the eighth aspect of the present invention, a kit is provided, which comprises the engineered TIL cells described in the first aspect of the present invention, the activated fusion protein described in the second aspect of the present invention, the retained fusion protein described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the nucleic acid construct described in the fifth aspect of the present invention, the vector described in the sixth aspect of the present invention, and / or the composition described in the seventh aspect of the present invention.

[0114] In another preferred embodiment, the kit comprises the engineered TIL cells described in the first aspect of the present invention, or a reagent for preparing the engineered TIL cells described in the first aspect of the present invention, wherein the reagent is selected from the following group:

[0115] (Y1) a polynucleotide encoding the activated fusion protein and the retained fusion protein; or

[0116] (Y2) A vector comprising: a polynucleotide encoding the activation-type fusion protein and / or a polynucleotide encoding the retention-type fusion protein.

[0117] In the ninth aspect of the present invention, provided is the use of the engineered TIL cells described in the first aspect of the present invention, the activated fusion protein described in the second aspect of the present invention, the retained fusion protein described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the nucleic acid construct described in the fifth aspect of the present invention, the vector described in the sixth aspect of the present invention, the composition described in the seventh aspect of the present invention, and / or the kit described in the eighth aspect of the present invention in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0118] In another preferred embodiment, provided is the use of the engineered TIL cells or the kit of the present invention in preparing a drug for treating tumors.

[0119] 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.

[0120] In the tenth aspect of the present invention, a method for treating a disease is provided, comprising administering the cell according to the first aspect of the present invention and / or the composition according to the seventh aspect of the present invention to a subject in need thereof.

[0121] In another preferred embodiment, the subject is a human or a mammal.

[0122] In another preferred embodiment, the disease is tumor or cancer.

[0123] 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.

[0124] 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

[0125] FIG1 shows a schematic diagram of the engineered TILs of the present invention killing tumor cells.

[0126] FIG2 shows a schematic diagram of the structures of the 055, 073, 076 and 083 constructs of the present invention.

[0127] Figure 3 shows the results of flow cytometry analysis of the positive rate of lentiviral-infected TILs, where TIL mock is a control TIL. The horizontal and vertical axes represent relative fluorescence intensity, and the trapezoids represent the proportion of positive cells.

[0128] FIG4 shows the detection results of TIL cell proliferation ability in each group, wherein TIL-MOCK is the control TIL.

[0129] FIG5 shows the detection results of TIL cell viability in each group, wherein TIL-MOCK is the control TIL.

[0130] Figure 6 shows the results of stem cell percentage analysis for each group of TIL cells, with TIL-MOCK serving as the control TIL. The horizontal and vertical axes represent relative fluorescence intensity, and the Q2 region indicates the proportion of cells that are both CD45RA and CD62L positive, indicating the proportion of stem T cells.

[0131] FIG7 shows the IFN-γ secretion detection results of TIL cells in each group, wherein Ctr MOCK is the control TIL.

[0132] FIG8 shows microscopic images of each group of TIL cells and tumor cells (HELA) co-cultured at a ratio of 3:1 for 24 hours, wherein TIL-MOCK is the control TIL.

[0133] FIG9 shows the results of the cell killing experiment of each group of TIL cells on tumor cells (HELA), wherein HELA only refers to tumor cells only, and TIL-MOCK refers to the control TIL.

[0134] FIG10 shows the results of cell killing experiments of TIL cells in each group on tumor cells (A-375 B2M KO).

[0135] Figure 11 shows the effects of the molecular switch of the present invention. The horizontal and vertical axes represent relative fluorescence intensity, and the (Q2+Q3) area represents the proportion of apoptotic cells. DETAILED DESCRIPTION

[0136] After extensive and in-depth research, the inventors have provided for the first time an engineered TIL cell, whose cell surface contains an exogenous membrane-bound DAP10-CD3ζ fusion protein, and whose endoplasmic reticulum or Golgi apparatus contains a retained fusion protein. The membrane-bound DAP10-CD3ζ fusion protein (activated fusion protein) of the present invention is expressed in TIL cells and can unexpectedly effectively initiate the activation of TIL cells, thereby more effectively killing tumor cells. Specifically, after the engineered TIL cells of the present invention recognize NKG2DL on the surface of tumor cells through NKG2D on their surface, they can further effectively activate TIL cell activation through the fusion protein of the present invention, thereby allowing TIL cells to effectively kill tumor cells. Experiments have shown that the retained fusion protein of the present invention can be effectively retained by the endoplasmic reticulum, thereby retaining the NKG2DL endogenously expressed by the TIL cells within the cells, avoiding mutual killing between TIL cells and preventing the occurrence of fragility during TIL cell culture, thereby significantly improving the viability of TIL cells. The engineered TIL cells of the present invention can be used to treat tumors. The present invention was completed on this basis.

[0137] DAP10

[0138] DAP10 is widely expressed in immune cells and is highly conserved throughout evolution. It exerts its unique intracellular signaling function by binding to cell receptors such as NKG2D. DAP10 transmits cell activation signals through its YINM motif, providing costimulatory signals in T cells.

[0139] The amino acid sequence of human DAP10 is accession number GenBank: AAD46986.1, and it has 92 amino acids, as shown at positions 1-92 in SEQ ID NO: 1. DAP is composed of multiple parts, including a signal peptide (positions 1-18), an extracellular domain (positions 19-48), a transmembrane domain (positions 49-69), and an intracellular domain (positions 70-92), wherein the intracellular domain contains a YINM motif.

[0140] The sequences of DAP10 in mammals (especially primates) are highly homologous and have similar functions. The homology between human and non-human primate DAP10 is as high as 90-100%, and the homology between human and rodent DAP10 is about 80%.

[0141] In the present invention, DAP10 includes wild-type and mutant DAP10, as long as the mutant DAP10 retains or substantially retains the function of wild-type DAP10, such as retaining ≥50% (preferably ≥60%, ≥70%, ≥80%, ≥90%, or even ≥100% (e.g., 100-200%)) of the wild-type DAP10 function. It should be understood that in the present invention, DAP10 also includes mutant proteins whose mutant function exceeds that of wild-type DAP10, for example, mutant DAP10 having ≥100% (e.g., 100-200%) of the wild-type DAP10 function.

[0142] In the present invention, the mutation of DAP10 may be naturally occurring or artificially introduced.

[0143] NKG2D and NKG2DL

[0144] NKG2D is an activating receptor expressed on the surface of NK cells, NKT cells, and CD8+ T cells. It plays a crucial role in innate immunity and is involved in the killing of tumor cells by various immune cells. The NKG2D ligand (NKG2DL) family of proteins primarily includes six cytomegalovirus UL16 binding proteins 1-6 and MHC class I-related molecules A and B (MICA and MICB). NKG2DL is largely absent in normal cells but is highly expressed on the cell surfaces of various tumors of diverse origin, including but not limited to colorectal cancer, liver cancer, and gliomas.

[0145] NKG2D is a DAP10-associated receptor, and the receptor complex is a hexamer composed of one NKG2D homodimer and two DAP10 homodimers.

[0146] After NKG2D binds to NKG2DL on the surface of tumor cells, the charged amino acid residues in the transmembrane region of the NKG2D homodimer connect to the TM residues of DAP10 through two salt bridges, forming a hexameric structure. This in turn induces phosphorylation of the YINM motif in the cytoplasm, which then activates the downstream phosphoinositide 3-kinase (PI3K) signaling pathway, transmitting activation signals. The signals generated by NKG2D can directly activate NK cells to exert a cytotoxic effect and act as a co-stimulatory signal to promote the activation of αβT cells and γδT cells and enhance their cytotoxic effect.

[0147] NK and NKT cells can kill tumor cells through NKG2D-NKG2DL binding, and CD8+ T cells can recognize and kill tumors that do not express MHC-I molecules through NKG2D-NKG2DL.

[0148] However, TIL cells themselves highly express NKG2D molecules, but because the NKG2D downstream molecule DAP10 only plays a co-stimulatory signal role in T cells, TIL cells cannot effectively kill tumor cells after recognizing tumor cells through NKG2D-NKG2DL due to the lack of the first signal of T cell activation.

[0149] In the present invention, an engineered TIL cell is provided that can be activated through the NKG2D-NKG2DL pathway to kill tumor cells.

[0150] DAP10 and CD3ζ fusion protein of the present invention

[0151] As used herein, the terms "activating fusion protein of the present invention," "membrane-bound DAP10-CD3ζ fusion protein of the present invention," and "DAP10 and CD3ζ fusion protein of the present invention" are used interchangeably and refer to the DAP10 and CD3ζ fusion protein of the present invention that has T cell activation function. The membrane-bound DAP10-CD3ζ fusion protein of the present invention has an extracellular structure, transmembrane domain, and intracellular domain derived from DAP10, and an intracellular structure derived from CD3, such as CD3ζ.

[0152] In the membrane-bound DAP10-CD3ζ fusion protein of the present invention, the transmembrane region can be the transmembrane region derived from DAP10, the transmembrane region of CD3, the transmembrane region derived from other membrane proteins, or an artificially synthesized transmembrane region.

[0153] In addition, in the membrane-bound DAP10-CD3ζ fusion protein of the present invention, the hinge region or additional linker region may or may not be contained between the extracellular structure and the transmembrane region, and the additional linker region may or may not be contained between the intracellular structure (such as CD3ζ) and the transmembrane region.

[0154] In a preferred embodiment, the extracellular structure and transmembrane region of the membrane-bound DAP10-CD3ζ fusion protein of the present invention are both derived from DAP10 protein, preferably from human DAP10 protein, such as from amino acids 1-69 of human DAP10 protein.

[0155] Typically, the activated fusion protein of the present invention includes the following elements:

[0156] (1) a DAP10 element or an active fragment thereof; and

[0157] (2) An intracellular signaling domain, which is derived from the intracellular segment of DAP10 and CD3ζ.

[0158] In another preferred embodiment, the membrane-bound DAP10-CD3ζ fusion protein of the present invention comprises (1) an extracellular structure, a transmembrane domain and an intracellular domain derived from the DAP10 element or its active fragment, and (2) an intracellular signaling domain derived from CD3ζ, which are connected in series from the N-terminus to the C-terminus.

[0159] In one embodiment, the intracellular signaling domain optionally further comprises a costimulatory molecule; the costimulatory molecule is selected from the group consisting of CD28, CD27, 4-1BB, CD40, and OX40.

[0160] The amino acid sequence of the preferred activated fusion protein of the present invention is as follows:

[0161] Among them, the signal peptide sequence is shown at positions 1-18 in SEQ ID NO:1, the sequence of the extracellular domain is shown at positions 19-48 in SEQ ID NO:1, the transmembrane domain sequence is shown at positions 49-69 in SEQ ID NO:1, the DAP10 intracellular domain sequence is shown at positions 70-92 in SEQ ID NO:1, and the CD3ζ sequence is shown at positions 93-206 in SEQ ID NO:1.

[0162] The membrane-bound DAP10-CD3ζ fusion protein of the present invention can serve as a signal for TIL cell activation. Specifically, after the NKG2D of TIL cells binds to the NKG2DL on the surface of tumor cells, the membrane-bound DAP10-CD3ζ fusion protein of the present invention can effectively activate TIL cells, enabling them to kill tumor cells.

[0163] The retention fusion protein of the present invention (tNKG2D-KDEL)

[0164] NKG2D ligand (NKG2DL) is not only highly expressed on the surface of tumor cells, but is also transiently expressed on activated TIL cells, allowing activated TIL cells to recognize and kill each other.

[0165] The present invention provides a retention-type fusion protein with a unique structure (referred to as "the retention-type fusion protein of the present invention"), which comprises a NKG2D extracellular domain element and a KDEL element connected in series.

[0166] The retention-type fusion protein of the present invention can retain endogenous NKG2DL produced by T cells (such as TIL cells) in the cell, thereby reducing the NKG2DL appearing on the surface of TIL cells, or even eliminating the NKG2DL appearing on the surface of TIL cells.

[0167] In the present invention, KDEL is Lys-Asp-Glu-Leu (also known as the KDEL signal sequence), which is typically located at the carboxyl terminus (C-terminus) of a protein. The KDEL sequence has a corresponding receptor on the Golgi membrane. Once it enters the Golgi, it is bound by the receptor on the Golgi, forming a reflux vesicle that is transported back to the endoplasmic reticulum. This KDEL sequence is referred to as an endoplasmic reticulum retention sequence.

[0168] Preferably, the present invention provides a retention-type fusion protein expressed in TIL cells, wherein the retention-type fusion protein fuses the extracellular region of NKG2D with the KDEL signal sequence to form a tNKG2D-KDEL protein.

[0169] After TIL cell activation, transiently expressed endogenous NKG2DL specifically binds to the extracellular domain of NKG2D in the retention-type fusion protein of the present invention, resulting in its retention in the endoplasmic reticulum. This prevents or eliminates the presence of the expressed endogenous NKG2DL on the TIL cell membrane. This effectively prevents mutual recognition and killing between TIL cells, reduces cell fragility, and thus improves TIL cell viability.

[0170] The amino acid sequence of a representative retention-type fusion protein of the present invention is shown below:

[0171] Among them, positions 22 to 156 in SEQ ID NO: 2 are the NKG2D extracellular region sequence; positions 157 to 160 are the KDEL sequence.

[0172] Preferably, the retention-type fusion protein of the present invention further comprises a signal peptide sequence, preferably a CD8a signal peptide, the sequence of which is MALPVTALLLPLALLLHAARP (SEQ ID NO: 3).

[0173] Nucleic acid constructs of the present invention

[0174] As used herein, the nucleic acid construct of the present invention can express the fusion protein and / or retention fusion protein of the present invention. The nucleic acid construct of the present invention comprises a sequence encoding the fusion protein and / or retention fusion protein of the present invention, and optionally comprises a hypoxia-responsive promoter.

[0175] The hypoxia-responsive promoter of the present invention is a promoter comprising n hypoxia-responsive elements HRE, i.e., n×HRE promoter. Hypoxia is a hallmark of solid tumors, and this unique environmental signal can be used for targeted cancer therapy, so that the target gene is highly expressed in the tumor microenvironment and not expressed or low expressed in normal tissues, thereby maximizing the function of the exogenous gene while reducing possible side effects. When constructing a nucleic acid molecule that expresses the fusion protein of the present invention, the present invention adopts 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 adopts a TK mini promoter containing n×HRE.

[0176] Preferably, the present invention uses the 3×HRE-TK-mini promoter.

[0177] 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.

[0178] 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 self-cleaving peptide is selected from: T2A, P2A, or a combination thereof.

[0179] 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.

[0180] 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.

[0181] Membrane-bound IL-15

[0182] The nucleic acid construct of the present invention preferably further comprises a third expression cassette that expresses membrane-bound IL-15 (mIL-15), i.e., a fusion protein comprising IL-15, a transmembrane domain, and an intracellular domain. In another preferred embodiment, the membrane-bound IL-15 is also linked to the first expression cassette via a cleavable linker peptide.

[0183] Interleukin IL-15 is a pro-survival cytokine that maintains long-lived CD8 + Memory T cell homeostasis, inhibition of activation-induced cell death (AICD), enhancement of in vivo anti-tumor activity and reversal of T cell anergy. Monomeric IL-15 is a small, unstable protein with a short serum half-life that requires supraphysiological administration to obtain an in vivo response. In the present invention, IL-15 is linked to a transmembrane domain and an intracellular domain to obtain membrane-bound IL-15, which has the function of maintaining the long-term persistence of the memory stem cell phenotype. Preferably, the intracellular domain is the CD86 intracellular domain, and preferably, the transmembrane domain is the CD86 transmembrane domain.

[0184] The membrane-bound IL-15 can increase the proportion of stem T cells and reduce the expression of T cell exhaustion molecules.

[0185] The membrane-bound IL-15 may further include a hinge region, a signal peptide and / or a linker. Preferably, the structural formula of the membrane-bound IL-15 is as shown in formula (I): X-IL-15-LH-TM-Cyto (I)

[0186] in,

[0187] “-” each independently represents no peptide or a connecting peptide;

[0188] X is none or signal peptide;

[0189] IL-15 is interleukin 15;

[0190] L is none or a linker;

[0191] H is a hinge region; the preferred hinge region is the CD86 hinge region;

[0192] TM is a transmembrane domain; the preferred transmembrane domain is the CD86 transmembrane domain;

[0193] Cyto is an intracellular domain, and the preferred intracellular domain is the CD86 intracellular domain.

[0194] The vector of the present invention

[0195] 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.

[0196] 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.

[0197] Engineered TIL cells of the present invention

[0198] 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. A schematic diagram of the TIL cells of the present invention recognizing and killing tumor cells is shown in Figure 1.

[0199] The engineered TIL cells of the present invention express the DAP10 and CD3ζ fusion protein of the present invention as an activation signal. After the NKG2D of the engineered TIL cells of the present invention binds to the NKG2DL on the surface of tumor cells, it can be successfully activated by the DAP10 and CD3ζ fusion protein to kill tumor cells.

[0200] In one embodiment, the engineered TIL cells of the present invention further express a retention fusion protein in which the extracellular region of NKG2D of the present invention is fused to the KDEL sequence. The endogenous NKG2DL briefly expressed on the surface of the TIL cells after activation specifically binds to the NKG2D region in the retention fusion protein, thereby being retained in the endoplasmic reticulum instead of being expressed on the TIL cell membrane, thereby preventing mutual recognition and killing between TIL cells, reducing cell fragility, and improving cell viability.

[0201] In one embodiment, the engineered TIL cells of the present invention further express the membrane-bound IL-15 of the present invention.

[0202] In one embodiment, the engineered TIL cells of the present invention further express a molecular switch element of the present invention. When the engineered TIL cells of the present invention present a safety risk, the molecular switch element can be used as a target to eliminate the risky cells using a corresponding drug. The molecular switch element of the present invention is selected from the group consisting of hEGFRt, BCMA, and CD20. Preferably, the molecular switch element of the present invention is hEGFRt.

[0203] Pharmaceutical composition of the present invention

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] The main advantages of the present invention include:

[0212] (1) After the NKG2D on the surface of the engineered TIL cells of the present invention recognizes the NKG2DL on the surface of the tumor cells, it can be effectively activated by the fusion protein of DAP10 and CD3ζ of the present invention, thereby enabling the TIL cells to effectively kill the tumor cells.

[0213] (2) The endogenous NKG2DL expressed by the engineered TIL cells of the present invention when activated can be retained in the cells by the retention fusion protein of the present invention, thereby avoiding mutual killing between TIL cells, preventing the occurrence of fragility during TIL cell culture, and improving the viability of TIL cells.

[0214] (3) The engineered TIL cells of the present invention can not only kill tumor cells by recognizing the antigen peptides presented by the MHC-I molecules of tumor cells through TCR, but also kill tumors through the NKG2D-NKG2DL pathway, thereby increasing the broad-spectrum recognition ability of traditional TIL cells for tumor cells, thereby enhancing the killing ability of TIL cells and effectively preventing the immune escape and recurrence of solid tumors.

[0215] (4) The engineered TIL cells of the present invention can also express a fusion protein of membrane-bound IL-15 containing the CD86 intracellular domain, thereby significantly improving the stemness and killing persistence of TIL cells.

[0216] (5) The engineered TIL cells of the present invention can be induced to express the fusion protein of the present invention under the hypoxia environment that is characteristic of solid tumors through a hypoxia-responsive promoter.

[0217] (6) The engineered TIL cells of the present invention can also express immune molecular switches, such as hEGFRt, avoiding the potential toxicity of continuous expression of exogenous proteins and improving the safety of clinical treatment.

[0218] 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 performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are by weight.

[0219] The sequence information involved in the following examples is shown in Table A:

[0220] Nucleotide or amino acid sequence of each element in Table A

[0221] Example 1: Preparation of TIL cells containing target genes

[0222] 1. Plasmid Construction

[0223] The CDS sequences of IL-15, DAP10, NKG2D, CD3ζ and the third and fourth domains of the extracellular segment of EGFR were obtained from NCBI, and some of the sequences were codon optimized.

[0224] As shown in Figure 2, four constructs, 055, 073, 076, and 083, were constructed. The molecular sequences of each construct were fully synthesized by GenScript, and the synthesized gene sequences were cloned into the basic vector pLV-EF1a-c-MYC-IRES-EGFP (purchased from Wuhan Miaoling) to obtain plasmids 055, 073, 076, and 083.

[0225] 2. Transformation and Plasmid Extraction

[0226] Remove the Trans109 competent cells from -80°C and quickly place them on ice. Once thawed, add the target plasmid (055, 073, 076, 083) and mix thoroughly. Let them rest on ice for 25 minutes. Heat shock the cells in a 42°C water bath for 45 seconds, then quickly return them to ice and let them rest for 2 minutes. Add 700 μl of sterile LB medium without antibiotics, mix thoroughly, and then recover the cells at 37°C, 200 rpm, for 70 minutes. Centrifuge at 5000 rpm for 1 minute to collect the cells, spread them onto LB medium containing the appropriate antibiotics, and incubate them in a 37°C incubator overnight. Pick a single colony and add it to 1 ml of LB medium containing antibiotics and incubate at 37°C, 200 rpm, for 2 hours. Expand this culture and culture overnight. Centrifuge 150 ml of the overnight culture at 5,000 g for 10 minutes, remove as much supernatant as possible, and collect the cells. Resuspend the pellet in 14 ml of solution P1 until thoroughly suspended. Add 14 ml of solution P2 to fully lyse the bacteria. Add 14 ml of pre-cooled solution P3 until a yellow flocculent precipitate appears, and centrifuge for 5 minutes. Collect the filtrate, add 14 ml of plasmid DNA binding solution to the filtrate, and mix well. Centrifuge for 2 minutes and remove the mixed solution. Add 10 ml of plasmid washing solution, centrifuge for 2 minutes, and discard the waste liquid. Add 10 ml of plasmid DNA washing solution 2 (add anhydrous ethanol), centrifuge for 2 minutes, and discard the waste liquid. Repeat the previous step. Remove residual ethanol and elute the plasmid DNA. Pass through an endotoxin removal column and centrifuge for 1 minute to elute the endotoxin-free plasmid DNA. Transfer the obtained plasmid to a 1.5 ml centrifuge tube.

[0227] 3. Lentivirus Preparation

[0228] The pLV-HRE-mIL-15 (055, 073, 076, 083) plasmids, psPAX2 vectors, and pMD2.G vectors containing the mIL-15 gene obtained in step 2 were purified separately; the plasmids were mixed with Lipo3000 in proportion and transfected into HEK-293T cells (cultured in 100 mm dishes) with a confluence of about 80%. 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. The mixture in centrifuge tube B was slowly dripped into centrifuge tube A, mixed, and allowed to stand at room temperature for 15-20 minutes.

[0229] 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.

[0230] 4. Lentiviral Titer Determination

[0231] 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.

[0232] 5. Preparation of TIL Cells

[0233] Place the tumor mass in a 10 cm culture dish and wash with PBS. Use sterile ophthalmic scissors or a scalpel to remove the necrotic area and connective tissue on the tumor mass, and then cut it into 1-3 mm pieces. 3Tumor fragments (e.g., lung cancer) are cultured in RPMI 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. Continue changing the medium if there is no gradual increase in lymphocytes or a decrease in cancer cells. 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 before harvesting TIL cells. Filter the collected cell suspension through a 40μm filter to remove tumor fragments. Centrifuge at 1000 rpm for 5 minutes, resuspend in an appropriate amount of REP complete medium, and set aside.

[0234] 6. Obtaining TIL cells after lentiviral infection

[0235] 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 medium, with 200 μl of the 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 hours of infection, the cells were collected and centrifuged at 1000 rpm, and the virus solution was discarded. An appropriate amount of REP complete medium (AIM V medium: A1640 medium = 1:1) was added to resuspend the cells, which were transferred to a 24-well plate and cultured in a 37°C, 5% CO2 incubator. After 72 hours of culture, the cells were collected.

[0236] Take 1×10 6 The above cells were transferred to a new 24-well plate and then placed in a hypoxic chamber (oxygen concentration of 1%) for hypoxia treatment (experimental group); the remaining TIL cells (as a non-hypoxic control group) were transferred to a new 24-well plate and cultured in a 37°C, 5% CO2 incubator. The TIL cells obtained by infecting the lentivirus containing the 055, 073, 076, and 083 constructs were named 055TIL, 073TIL, 076TIL, and 083TIL, respectively.

[0237] Example 2: Flow cytometry detection of lentivirus-infected TIL positive rate

[0238] Take the various cells prepared in Example 1 and divide each group into two parts: a negative group and a test group. The cell volume of each group is 50 μl, i.e., 2.5×10 5cells; IL-15 antibody (Beijing Baixinyi Biotechnology Co., Ltd., A09D21-9E) was 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) 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; 1 ml of PBS containing 2% FBS was added and centrifuged at 400g for 5 minutes; the supernatant was discarded, and 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 tested on the machine.

[0239] The test results are shown in Figure 3. Flow cytometry data showed that the positive rates of 055, 073, 076, and 083 TILs were 30.8%, 24.5%, 27.7%, and 32.7%, respectively, all showing high positive rates. Among them, the positive rate of 083 TIL (32.7%) was unexpectedly higher than that of other TILs.

[0240] Example 3: Detection of proliferation ability and viability of each TIL cell

[0241] Each group of TIL cells and trophoblast cells (IL-21NK cell expansion reagent, Zhongying Bio, product number ZY-NKZ-0104) were cultured at a ratio of 1:25, and the cell density was adjusted to 5×10 5 Cells were cultured in REP medium (RPMI 1640:AIM-V=1:1) in 6-well plates at 2 ml / well. Cell counts were performed every two days. Fresh medium was added to adjust the cell density to 5×10 5 The cells were cultured for 14 days, and the cell count, viability and stemness were determined.

[0242] The results of the proliferation capacity test are shown in Figure 4. The proliferation folds of the control TIL, 055TIL, 073TIL, 076TIL, and 083TIL after 14 days of culture were 220, 203, 57, 167, and 198, respectively. The proliferation capacity of 055TIL and 083TIL was basically the same as that of the control TIL, the proliferation fold of 076TIL was slightly reduced, and the proliferation fold of 073TIL was significantly reduced, far lower than the proliferation folds of TIL in other groups.

[0243] The results of cell viability testing are shown in Figure 5. The viabilities of the control TIL, 055 TIL, and 083 TIL were very high, at 95%, 94%, and 94%, respectively. The viability of 076 TIL was slightly reduced to 85%. The viability of 073 TIL was significantly reduced, and cell expansion was severely affected, with a viability of only 71%.

[0244] Example 4: Detection of the Stem Cell Ratio of Each TIL Cell

[0245] Take each group of cells prepared in Example 1 and divide each group into two parts: a negative group and a test group. The cell volume of each group is 50 μl, i.e., 2.5×10 5 cells; 50 μl of Alexa Fluor 500 μl prepared in PBS containing 2% FBS was added to the test group. 700 anti-human CD45RA antibody (BioLegend, 304120), APC anti-human CD62L antibody (BioLegend, 304810), and antibody mixture were incubated at room temperature for 8 minutes; 50 μl of 7-AAD mixture prepared with PBS containing 2% FBS was added to the cells in 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 the cells were 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 tested.

[0246] The test results are shown in Figure 6. The proportion of CD45RA and CD62L double-positive cells was used to indicate the proportion of stem T cells. Compared with uninfected TIL cells (control TIL), the proportion of stem cells increased from 6.37% to 11% (055TIL), 10.3% (076TIL), and 9.71% (083TIL), respectively.

[0247] This indicates that the membrane-bound IL-15 construct of the present invention significantly increases the proportion of stem T cells in TIL cells. In addition, the inclusion of the membrane-bound DAP10-CD3ζ fusion protein has little effect on the proportion of stem T cells.

[0248] Example 5: Detection of IFN-γ secretion of each TIL cell

[0249] TILs were co-cultured with NKG2DL-high-expressing cells, such as CALU6, HELA, and HCC827, at a ratio of 3:1. Supernatants were collected and assayed using an IFN-γ ELISA kit (Human IFN-γ Pre-made ELISA Kit, Dakoway, 2307-3) according to the kit instructions: Sample addition: Add diluted cytokine standards to the standard wells at 100 μl / well. Dilute the samples with 1× Dilution Buffer R and add them to the sample wells at 100 μl / well. Cover with a film sealer and incubate at room temperature for 2 hours. Wash: Remove the liquid from the wells and add 1× Wash Buffer (300 μl / well). Allow to stand for 1 minute, then discard the liquid. Repeat three times, blot dry on filter paper each time. Add detection antibody: Add biotinylated antibody working solution at 100 μl / well. Cover with a film sealer and incubate at room temperature for 1 hour. Wash the plate repeatedly. Add enzyme: Add streptavidin-HRP working solution, 100 μl / well. Cover with film and incubate at room temperature (18-25°C) for 30 minutes. Repeat washing. Color development: Add TMB, 100 μl / well, incubate at room temperature, protected from light, for 5-30 minutes. Determine the termination of the reaction based on the depth of the color in the well (dark blue). Generally, 10-20 minutes of color development is sufficient to achieve good results. Stop the reaction: Quickly add stop solution, 100 μl / well, to terminate the reaction. Read the OD value at 450 nm on a microplate reader. Analyze the data using the four-parameter method.

[0250] The results are shown in Figure 7 and Tables 1-3. After co-culture with various tumor cells, the secretion of IFN-γ by 076TIL and 083TIL was much higher than that by uninfected control TIL and 055TIL.

[0251] Table 1 IFN-γ secretion amount (pg / ml) of each group of TIL molecules co-cultured with CALU6 cells

[0252] Table 2 IFN-γ secretion of each group of TIL molecules co-cultured with HELA cells (pg / ml)

[0253] Table 3 IFN-γ secretion amount of each group of TIL molecules co-cultured with HCC827 cells (pg / ml)

[0254] The above results show that compared with 055TIL, which only recognizes tumor cells through TCR, the 076TIL and 083TIL expressing the membrane-bound DAP10-CD3ζ fusion protein of the present invention can not only use TCR to recognize tumor cells, but also more efficiently activate TIL cells through the NKG2D-NKG2L pathway. At the same time, 083 prevents the fragility of TIL through endoplasmic reticulum retention technology, making the activity rate of 083TIL higher than 076TIL, and it is more easily activated during co-culture with target cells, thereby showing a higher IFN-γ secretion.

[0255] Example 6: In vitro killing test results of each TIL

[0256] Take 1×10 5 Calu-6 tumor cells (ATCC, HTB-56 TM )、HELA(ATCC,CRM-CCL-2 TM ), HCC827 (CRL-2868) and A-375 B2M KO (Guangzhou Yuanjing, catalog number: YKO-H486) were seeded in 24-well plates. After 24 hours, TIL cells were diluted to a density of 1.5×10 6 Aspirate the culture medium from the original wells of the 24-well plate. Add 1 ml of TIL cell culture medium to each well. After 24 hours, aspirate the culture medium from the wells, centrifuge, and use the supernatant for ELISA analysis. Wash the cells in the wells with PBS, and assay tumor cell viability using the CCK8 assay.

[0257] After co-culture of TIL and tumor cells (HELA) in each group at a ratio of 3:1 for 24 hours, microscopic observation and photography were performed. The results are shown in Figure 8: In the HELA alone and HELA and control TIL co-culture experimental groups, HELA cells grew well, growing in a dense monolayer. In the HELA+055TIL experimental group, HELA cells grew slowly, failed to fill the cell culture well, and some cells became rounded and fell off. In the HELA+076TIL and 083TIL experimental groups, HELA cells became rounded and fell off over a large area.

[0258] The results of the cytotoxicity test of each group of TIL against HELA tumor cells at E:T=3:1 are shown in Figure 9 and Table 4. Compared with the control TIL and 055TIL, the cytotoxicity of 076TIL and 083TIL against tumor cells was significantly improved, increasing by about 3 times.

[0259] Table 4 The percentage of live target cells after HELA cells were killed by different TILs (%)

[0260] The killing effects of TIL in each group on A-375 B2M KO tumor cells at E:T ratios of 1:1, 3:1, and 5:1 are shown in Figure 10 and Table 5. The modified 076 and 083 TILs had a significant killing effect on the melanoma cell line A375 (B2M KO) compared to 055 TIL and unmodified control TIL in a dose-dependent manner. Under conditions of high-efficiency target ratio, the killing was more obvious, with a cytotoxicity of about 60% (100% minus the proportion of live cells).

[0261] Table 5 The percentage of live target cells after A-375 B2MKO was killed by different TILs (%)

[0262] Example 7: Antibody-dependent cellular cytotoxicity assay (ADCC)

[0263] ADCC (antibody dependent cell-mediated cytotoxicity) refers to the binding of an antibody's Fab fragment to an antigenic epitope on the surface of a target cell, and the binding of its Fc fragment to FcRs on killer cells (NK cells, macrophages, neutrophils, etc.), mediating direct killing of target cells by these cells. EGFR binds to cetuximab, leading to target cell death through ADCC. Rituximab does not bind to EGFR and does not induce cell death through ADCC, so it serves as a control to demonstrate the specificity of the design.

[0264] NK cells were isolated and expanded from the peripheral blood of healthy volunteers using an NK cell culture kit (purchased from Fujian Sany Hematopoietic Technology Co., Ltd., CT-001) according to the instructions and labeled with CytoTell Blue. Target cells (TILs infected with 076LV) were resuspended in culture medium and the cell density was adjusted to 5×10 5 cells / ml, 1 ml was taken and added to three wells of a 6-well plate. Cetuximab (Merckelion Pharmaceuticals, Germany) and rituximab (Rituximab, Roche Diagnostics GmbH, H0334) were added to a concentration of 200 μg / ml, and the same volume of PBS was added. After mixing, the cells were incubated at room temperature for 40 minutes. The NK cells were resuspended in culture medium and the cell density was adjusted to 5×10 6 1 ml of each solution was added to the three wells of a 6-well plate to achieve an effector-target ratio of 10:1. The final concentration of cetuximab and rituximab in the system was 100 μg / ml, and the total volume was 2 mL. The system was mixed and incubated in an incubator for 24 hours. After 24 hours, changes in cell apoptosis were detected using a PE-coupled Annexin-V apoptosis detection kit (BD Biosciences, 559763).

[0265] The results are shown in Figure 11. When 076 TIL was added to NK cells and rituximab (which recognizes CD20) was added simultaneously, the apoptosis rate ranged from 5.5% to 8.1%, a relatively low level. When 076 TIL was added to the presence of NK cells and cetuximab (which recognizes EGFR), the apoptosis rate was approximately 30%. These experimental results demonstrate that hRGFRt can act as a specific molecular switch, improving the safety of clinical use.

[0266] discuss

[0267] The present inventors have provided for the first time a novel and unique membrane-bound DAP10 and CD3ζ fusion protein. The membrane-bound DAP10-CD3ζ fusion protein of the present invention can unexpectedly effectively activate TIL cells, thereby more effectively killing tumor cells.

[0268] In the present invention, after the engineered TIL cells recognize NKG2DL on the surface of tumor cells through the NKG2D on their surface, the TIL cells can be further effectively activated by the fusion protein of the present invention, thereby enabling the TIL cells to effectively kill tumor cells.

[0269] Taking 083TIL as an example, it utilizes a hypoxia-inducible promoter to regulate the expression of the present invention's DAP10 and CD3ζ fusion protein. Once TIL cells reach tumor tissue or its surroundings, they express the membrane-bound DAP10-CD3ζ fusion protein of the present invention. Upon recognition of tumor cells via NKG2D-NKG2DL, they initiate T activation through DAP10 and CD3ζ functional elements, effectively killing tumor cells.

[0270] The modified TIL cells of the present invention can not only kill tumor cells by recognizing tumor cell MHC-I molecules and presenting antigen peptides through TCR, but also kill tumors through the NKG2D-NKG2DL pathway, which can effectively prevent the immune escape and recurrence of solid tumors.

[0271] In addition, the present invention also provides a retention-type fusion protein with a novel and unique structure. Experiments have shown that the retention-type fusion protein of the present invention can be effectively retained by the endoplasmic reticulum, thereby retaining NKG2DL endogenously expressed by TIL cells in the cells, avoiding mutual killing between TIL cells, preventing the occurrence of fragility during TIL cell culture, and thus significantly improving the viability of TIL cells.

[0272] In addition, the engineered TIL cells of the present invention can further express a novel and unique membrane-bound IL-15, thereby further improving the stemness of the TIL cells.

[0273] In addition, the engineered TIL cells of the present invention can further express the hEGFRt molecular switch (such as 076TIL), which can specifically cause apoptosis of related TILs through ADCC, ensuring the clinical safety of TIL therapy. At the same time, cetuximab can also be used for flow cytometry detection as a positive rate indicator.

[0274] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. 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 claims appended hereto.

Claims

1. An engineered TIL cell with enhanced tumor recognition and killing ability, characterized in that, The engineered TIL cells express an exogenous fusion protein selected from the group consisting of an activating fusion protein, a retention fusion protein, or a combination thereof; wherein the activating fusion protein is a membrane-bound DAP10-CD3ζ fusion protein; the retention fusion protein comprises an extracellular domain of NKG2D and a KDEL sequence.

2. The engineered TIL cells according to claim 1, wherein, The activating fusion protein comprises: (1) an extracellular domain that contains a DAP10 element or an active fragment thereof; (2) a transmembrane domain; and (3) an intracellular signaling domain that contains the intracellular domain of DAP10 and CD3ζ; wherein the extracellular domain and the intracellular signaling domain are tandemly linked together by the transmembrane domain.

3. The engineered TIL cells according to claim 2, wherein, The intracellular signaling domain further comprises a co-stimulatory domain.

4. The engineered TIL cells according to claim 1, wherein, The amino acid sequence of the activating fusion protein is as shown in SEQ ID NO:

1.

5. The engineered TIL cells according to claim 1, wherein, The amino acid sequence of the retention fusion protein is as shown in SEQ ID NO:

2.

6. The engineered TIL cells according to claim 1, wherein The engineered TIL cells have the activating fusion protein on the cell membrane; and / or the engineered TIL cells have the retention fusion protein in the endoplasmic reticulum or Golgi apparatus.

7. The engineered TIL cells according to claim 1, wherein, The engineered TIL cells contain a polynucleotide that encodes the activating fusion protein and / or the retention fusion protein.

8. The engineered TIL cells according to claim 1, wherein, The engineered TIL cells contain a nucleic acid construct that comprises a first expression cassette, and the first expression cassette comprises a polynucleotide encoding the activating fusion protein and / or a polynucleotide encoding the retention fusion protein.

9. The engineered TIL cells according to claim 8, wherein, The first expression cassette further contains a promoter selected from the group consisting of a constitutive promoter, an inducible promoter, or a combination thereof.

10. The engineered TIL cells according to claim 1, characterized in that, The engineered TIL cells contain a vector that comprises a polynucleotide encoding the activating fusion protein and / or a polynucleotide encoding the retention fusion protein.

11. The engineered TIL cells according to claim 1, wherein, The engineered TIL cells further express a membrane-bound IL-15 fusion protein, which comprises the following elements: (i) interleukin 15 or a functionally active fragment thereof; (ii) a transmembrane domain; and (iii) an intracellular domain; wherein the intracellular domain is the intracellular domain of CD86.

12. A composition, characterized in that, The composition comprises the engineered TIL cells according to claim 1 and a pharmaceutically acceptable carrier.

13. A kit, characterized in that, The kit comprises the engineered TIL cells according to claim 1, or reagents for preparing the engineered TIL cells according to claim 1, wherein the reagents are selected from the group consisting of: (Y1) a polynucleotide that encodes the activating fusion protein and the retention fusion protein; or (Y2) a vector that comprises a polynucleotide encoding the activating fusion protein and / or a polynucleotide encoding the retention fusion protein.

14. Use of the engineered TIL cells according to claim 1, or the kit according to claim 13, in the preparation of a medicament for treating a tumor.

15. A method for treating a disease, characterized in that, The method comprises administering the cells according to claim 1 and / or the composition according to claim 12 to a subject in need.

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