Engineered TIL cells with enhanced serial killing ability and their applications

Abstract

The present invention relates to an engineered TIL cell with enhanced continuous killing ability and its application. Specifically, the present invention relates to an engineered TIL cell expressing a long-acting activation element, whose cell surface contains a modified DAP10 fusion protein, which can effectively start the activation of TIL cells and reduce the exhaustion phenomenon caused by excessive activation of TIL. The engineered TIL cells of the present invention can recognize tumor cells through the NKG2D-NKG2DL pathway and reduce the escape of tumor cells; in addition, during the culture stage, the engineered cells of the engineered TIL cells of the present invention have excellent amplification ability and cell viability. The engineered TIL cells of the present invention can kill tumor cells efficiently and durably, and have application prospects in the field of tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to engineered TIL cells with enhanced continuous killing ability and applications thereof. Background Art

[0002] Adoptive cellular immunotherapy (ACI or AIT) refers to the transfer of immune cells with anti-tumor activity to cancer patients, especially genetically engineered immune cells, to achieve direct killing of tumor cells or to kill tumor cells by stimulating the body's immune response. Currently, the most widely used cell immunotherapies include CAR-T, TCR-T, CAR-NK, etc. TIL cells, or tumor-infiltrating lymphocytes, play an important role in the body's specific immune response to tumor cells and have outstanding advantages over other immune cells.

[0003] First, tumor-infiltrating lymphocytes (TILs) can recognize multiple tumor-specific neoantigens and tumor-associated antigens, making them more effective in dealing with tumor heterogeneity; second, TILs usually contain certain effector memory T cells, which express chemokine receptors after being stimulated by tumor antigens in vivo, making them easier to localize in tumor tissues after reinfusion; in addition, TILs are derived from the patient himself and have lower toxicity. Therefore, TIL therapy has shown great potential in the field of solid tumors.

[0004] However, after activation, TIL cells derived from tumor tissues are often in a terminally differentiated and exhausted state, resulting in a weakened ability of TIL cells to kill tumor cells; most of the TIL cells cultured in vitro are non-tumor-specific bystander cells. How to transform bystander cells into effector cells with the ability to recognize and kill tumor cells to improve the therapeutic effect of TIL cells is one of the problems to be solved in the field of genetically engineered TIL. In addition, tumor cells often escape the killing of immune cells by reducing the expression of MHC-I molecules or TAP gene mutations, resulting in loss of antigen presentation function.

[0005] Therefore, there is an urgent need in the art to develop engineered TIL cells with efficient and durable tumor cell killing capabilities. Summary of the invention

[0006] The purpose of the present invention is to provide engineered TIL cells with efficient and lasting tumor cell killing ability.

[0007] In the first aspect of the present invention, an engineered TIL cell is provided, wherein the engineered TIL cell expresses an exogenous long-acting activated protein, wherein the long-acting activated protein comprises a DAP10 element and a long-acting activated element, and the sequence of the long-acting activated element is shown in SEQ ID NO:2.

[0008] In another preferred embodiment, the long-acting activated protein includes the following structures fused together from the N-terminus to the C-terminus:

[0009] (1) an extracellular domain, wherein the extracellular domain comprises a DAP10 element or an active fragment thereof;

[0010] (2) a transmembrane domain; and

[0011] (3) an intracellular signaling domain, wherein the intracellular signaling domain comprises a DAP10 intracellular domain and a long-acting activation element.

[0012] In another preferred embodiment, the long-acting activated protein has a structure as shown in Formula I:

[0013] L-ECD-TM-ICD1-ICD2-ICD3 (Formula I)

[0014] In the formula,

[0015] “-” each independently represents a peptide bond or a connecting peptide;

[0016] L is none or signal peptide;

[0017] ECD is the extracellular domain;

[0018] TM is the transmembrane domain

[0019] ICD1 is the intracellular domain from the DAP10 protein;

[0020] ICD2 has no or costimulatory domain;

[0021] ICD3 is a long-acting activation element.

[0022] In another preferred embodiment, the L is none.

[0023] In another preferred embodiment, the L is a signal peptide selected from the following group of proteins: DAP10, GM-SCF receptor α, CD8, and CD28.

[0024] In another preferred embodiment, the amino acid sequence of L is as shown in positions 1-18 of SEQ ID NO:1.

[0025] In another preferred embodiment, the ECD is from the extracellular domain of DAP10 protein.

[0026] In another preferred embodiment, the amino acid sequence of the ECD is shown in positions 19-48 of SEQ ID NO:1.

[0027] In another preferred embodiment, the TM is a transmembrane domain from DAP10, CD8, or CD28.

[0028] In another preferred embodiment, the TM is a transmembrane domain from DAP10.

[0029] In another preferred embodiment, the amino acid sequence of the TM is shown in positions 49-69 of SEQ ID NO:1.

[0030] In another preferred embodiment, the ICD1 is the intracellular domain of DAP10 protein.

[0031] In another preferred embodiment, the sequence of ICD1 is shown in positions 70-92 of SEQ ID NO:1.

[0032] In another preferred embodiment, the ICD2 is absent.

[0033] In another preferred embodiment, the ICD2 is one, two or more co-stimulatory domains derived from a protein selected from the group consisting of CD40, CD27, 4-1BB, OX40, or a combination thereof.

[0034] In another preferred embodiment, the ICD2 is a co-stimulatory domain from CD40 protein.

[0035] In another preferred example, the sequence of ICD2 is shown as SEQ ID NO:15.

[0036] In another preferred example, the amino acid sequence of ICD3 is shown in SEQ ID NO:2.

[0037] In another preferred example, the amino acid sequence of the long-acting activated protein is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0038] In another preferred embodiment, the engineered TIL cells have the long-acting activated protein on their cell membrane.

[0039] In another preferred embodiment, the engineered TIL cells have one or more properties selected from the following group:

[0040] (1) Long-lasting tumor cell-killing ability;

[0041] (2) reduce the exhaustion of TIL cells;

[0042] (3) Reduce tumor immune escape and recurrence;

[0043] (4) Improved in vivo expansion capability.

[0044] In another preferred embodiment, the activation is self-activation, that is, the engineered TIL cells expressing the long-acting activation protein are activated.

[0045] In another preferred example, the activated engineered TIL cells can effectively kill target cells through the interaction of NKG2D-NKG2DL.

[0046] In another preferred embodiment, the engineered TIL cells contain a polynucleotide encoding the long-acting activated protein.

[0047] In another preferred embodiment, the polynucleotide is DNA, RNA, or a combination thereof.

[0048] In another preferred embodiment, the engineered TIL cells contain a vector, and the vector comprises: a polynucleotide encoding the long-acting activated protein.

[0049] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0050] In another preferred embodiment, the viral vector includes: a lentiviral vector, an adenoviral vector, and a yellow fever virus vector.

[0051] In another preferred embodiment, the vector is a plasmid.

[0052] In another preferred embodiment, the engineered TIL cells also express a membrane-bound IL-15 fusion polypeptide, which includes the following elements fused together:

[0053] (i) interleukin 15 (IL-15);

[0054] (ii) CD86 transmembrane domain and CD86 intracellular domain.

[0055] In another preferred embodiment, the amino acid sequence of IL-15 is shown in SEQ ID NO:7.

[0056] In another preferred embodiment, the element (i) and the element (ii) directly or optionally comprise a linker and / or a hinge region.

[0057] In another preferred embodiment, the amino acid sequence of the linker is as shown in SEQ ID NO:8.

[0058] In another preferred embodiment, the amino acid sequence of the hinge region is shown in SEQ ID NO:9.

[0059] In another preferred example, the amino acid sequences of the CD86 transmembrane domain and intracellular domain are as shown in SEQ ID NO:10.

[0060] In another preferred example, the N-terminus of the fusion polypeptide optionally comprises a signal peptide, and the amino acid sequence of the signal peptide is shown in SEQ ID NO:6.

[0061] In another preferred embodiment, the engineered TIL cells contain a nucleic acid construct, which comprises a polynucleotide encoding the long-acting activated protein.

[0062] In another preferred embodiment, the nucleic acid construct contains a promoter, and the promoter is selected from the following group: a constitutive promoter, an inducible promoter, or a combination thereof.

[0063] In another preferred embodiment, the promoter is a hypoxia-responsive promoter.

[0064] In another preferred embodiment, the hypoxia-responsive promoter is a promoter comprising n hypoxia response elements HRE, that is, a promoter containing n×HRE elements, where n is an integer selected from 1-20.

[0065] 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.

[0066] In another preferred embodiment, the hypoxia-responsive promoter is selected from the following group: TK-mini promoter containing n×HRE elements, CMV-mini promoter containing n×HRE elements, IL2-mini promoter containing n×HRE elements.

[0067] In another preferred embodiment, the hypoxia-responsive promoter is a 3×HRE TK-mini promoter.

[0068] In another preferred example, the nucleic acid construct further comprises a nucleotide sequence encoding the membrane-bound IL-15 fusion polypeptide.

[0069] 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.

[0070] In another preferred embodiment, the molecular switch element is hEGFRt.

[0071] In another preferred embodiment, the nucleotide sequence encoding the long-acting activated protein, the nucleotide sequence encoding the membrane-bound IL-15 fusion polypeptide, and the nucleotide sequence encoding the molecular switch element are connected by a cleavable connecting peptide coding sequence.

[0072] In another preferred embodiment, the cleavable connecting peptide is a self-cleaving 2A peptide, preferably a T2A peptide.

[0073] In another preferred embodiment, the nucleotide sequence encoding the long-acting activated protein, the nucleotide sequence encoding the membrane-bound IL-15 fusion polypeptide, and the nucleotide sequence encoding the molecular switch element are each independently located in an expression cassette.

[0074] In the second aspect of the present invention, a pharmaceutical composition is provided, comprising the engineered TIL cells as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0075] In a third aspect of the present invention, a kit is provided, the kit comprising the engineered TIL cells as described in the first aspect of the present invention, or a reagent for preparing the engineered TIL cells as described in the first aspect of the present invention, wherein the reagent is selected from the following group:

[0076] (Y1) a polynucleotide encoding the long-acting activated protein; or

[0077] (Y2) A vector comprising: a polynucleotide encoding the long-acting activated protein.

[0078] In a fourth aspect of the present invention, there is provided a use of the engineered TIL cells as described in the first aspect of the present invention in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0079] In another preferred embodiment, the tumor includes: a solid tumor, a blood tumor, or a combination thereof.

[0080] In another preferred embodiment, the tumor is selected from the following group: lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

[0081] In the fifth aspect of the present invention, a method for treating a disease is provided, the method comprising administering the cell as described in the first aspect of the present invention and / or the pharmaceutical composition as described in the second aspect of the present invention to a subject in need thereof.

[0082] In another preferred embodiment, the subject is a human or a mammal.

[0083] In another preferred embodiment, the disease is a tumor or cancer.

[0084] In another preferred embodiment, the tumor includes: a solid tumor, a blood tumor, or a combination thereof.

[0085] In another preferred embodiment, the tumor is selected from the following group: lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

[0086] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 A schematic diagram of the construction of the 076, 095, and 096 molecules is shown.

[0088] Figure 2 The results of TIL cell infection positivity test in each group are shown.

[0089] Figure 3 The results of TIL cell proliferation ability detection in each group are shown.

[0090] Figure 4 The results of cell viability detection of each group of TIL cells after 9 days of expansion culture are shown.

[0091] Figure 5 The results of the secretion of cytokine IFN-γ by co-culture of TIL cells and tumor cells in each group are shown.

[0092] Figure 6 A schematic diagram of the experimental design for testing the serial killing ability of TIL cells on tumor cells is shown.

[0093] Figure 7 Microscope observations showing the results of continuous killing of tumor cells by TIL cells in each group.

[0094] Figure 8 The results of cytotoxicity detection during the continuous killing of tumor cells by TIL cells in each group are shown. DETAILED DESCRIPTION

[0095] After extensive and in-depth research, the inventors have provided for the first time an engineered TIL cell, whose cell surface contains a modified DAP10 fusion protein, and the fusion protein contains a long-acting activation element of the present invention obtained through a large number of screenings, which can unexpectedly effectively start the activation of TIL cells and reduce the exhaustion phenomenon caused by excessive activation of TIL. The engineered TIL cells of the present invention can recognize tumor cells through the NKG2D-NKG2DL pathway, reduce the escape of tumor cells, and be persistently activated to start killing ability, thereby achieving efficient and persistent killing of tumor cells. The present invention was completed on this basis.

[0096] Activation of TIL cells

[0097] Natural TIL cells are activated after specifically recognizing tumor cells through MHC-I molecules on the surface of tumor cells, and have the ability to specifically kill tumor cells. However, among the TIL cells obtained by in vitro culture, most of the cells are non-tumor-specific bystander cells and cannot be activated well. In addition, tumor cells often escape the killing of TIL cells by reducing the expression of MHC-I molecules. In the present invention, TIL cells are engineered so that TIL cells can be activated through the NKG2D-NKG2DL pathway, thereby enhancing the ability of TIL cells to recognize and kill tumor cells and reducing the escape of tumor cells.

[0098] NKG2D is an activating receptor expressed on the surface of NK cells, NKT cells, and CD8+T cells. It plays an important role in natural immunity and is involved in the killing of tumor cells by various immune cells. NKG2D ligand (NKG2DL) is basically not expressed in normal cells, but has high levels of expression on the surface of various tumor cells from different sources (such as colorectal cancer, liver cancer, brain glioma, etc.). NK and NKT cells can directly recognize and bind to each other through the NKG2D-NKG2DL form, thereby killing tumor cells.

[0099] However, in TIL cells, the downstream molecule DAP10 of NKG2D only plays a role of costimulatory signal. After TIL cells recognize tumor cells through NKG2D-NKG2DL, they cannot effectively kill tumor cells due to the lack of the first activation signal. Therefore, in the present invention, TIL cells are engineered to enable them to recognize and kill tumor cells through the NKG2D-NKG2DL pathway.

[0100] After activation, TIL cells derived from tumor tissues are often in a terminally differentiated and exhausted state, which limits the ability of TIL cells to continuously kill tumor cells. In addition, the engineered TIL cells available in the prior art are prone to being in an over-activated state and are prone to obvious exhaustion during the killing process, which is not conducive to the long-term and effective killing of tumor cells by TIL cells.

[0101] In the present invention, a long-acting activated engineered TIL cell is provided, which expresses the long-acting activated protein of the present invention and has an excellent function of continuously killing tumor cells.

[0102] DAP10

[0103] DAP10 exerts its unique intracellular signal transduction function by binding to cell receptors such as NKG2D. It is highly conserved during evolution. 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%.

[0104] The amino acid sequence accession number of human DAP10 is GenBank: AAD46986.1, and it has 92 amino acids, as shown in SEQ ID NO: 1:

[0105] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSSCSGCGSLSLPLLA GLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRG (SEQ ID NO: 1).

[0106] The signal peptide of DAP10 is at positions 1-18 of SEQ ID NO: 1, the extracellular domain is at positions 19-48, the transmembrane domain is at positions 49-69, and the intracellular domain is at positions 70-92.

[0107] In the present invention, DAP10 includes wild-type and mutant DAP10, as long as the mutant DAP10 retains or substantially retains the function of the wild-type DAP10, such as retaining ≥50% (preferably ≥60%, ≥70%, ≥80%, ≥90%) of the wild-type DAP10 function. It should be understood that in the present invention, DAP10 also includes mutant proteins whose mutant functions exceed those of the wild-type DAP10, such as mutant DAP10 having ≥100% (such as 100-200%) of the wild-type DAP10 function. In the present invention, the mutation of DAP10 may be naturally occurring or artificially introduced.

[0108] Long-acting activated protein of the present invention

[0109] As used herein, the term "long-acting activation protein of the present invention" refers to a fusion protein having TIL cell activation function that contains a DAP10 element and a long-acting activation element of the present invention.

[0110] In the present invention, the "long-acting activation element" is a sequence obtained through screening that has a signal transmission function, and its amino acid sequence is shown in SEQ ID NO: 2:

[0111] GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 2).

[0112] In the present invention, the long-acting activation element also includes a sequence having sequence identity with SEQ ID NO: 2, for example, a sequence having ≥85% (e.g., ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity with SEQ ID NO: 2, and a sequence having the same or similar TIL cell activation function as the sequence shown in SEQ ID NO: 2.

[0113] The long-acting activated protein of the present invention is a membrane-bound fusion protein, comprising a DAP10 element and a long-acting activated element, specifically comprising the extracellular domain, transmembrane domain, intracellular domain and long-acting activated element of DAP10, and optionally comprising a signal peptide, and optionally comprising one or more additional co-stimulatory domains.

[0114] In one embodiment, the long-acting activated protein of the present invention contains a signal peptide. The signal peptide is a polypeptide located in the fusion protein N, which plays a guiding role in the processes of protein folding, transport and positioning. In the present invention, the selection of signal peptides is not particularly limited, including signal peptides commonly used in the art, such as: DAP10 signal peptide, GM-SCF receptor α signal peptide, CD8 signal peptide, CD28 signal peptide, etc., but not limited thereto. In one embodiment, the signal peptide is a DAP10 signal peptide, and its sequence is shown in positions 1-18 of SEQ ID NO:1. In one embodiment, the sequence of the signal peptide is shown in SEQ ID NO:12.

[0115] In one embodiment, the long-acting activated protein of the present invention does not contain a signal peptide. During the protein maturation process, the signal peptide is generally removed, so the long-acting activated protein that has been localized on the cell membrane does not contain a signal peptide.

[0116] The extracellular domain of the long-acting activated protein of the present invention is an extracellular domain from a DAP10 protein, preferably human DAP10. In one embodiment, the sequence of the extracellular domain is as shown in positions 19-48 of SEQ ID NO:1.

[0117] In the present invention, the transmembrane domain of the long-acting activated protein is not particularly limited, and is preferably a transmembrane domain from DAP10, CD8, or CD28, and more preferably a transmembrane domain from DAP10. In one embodiment, the sequence of the transmembrane domain is as shown in positions 49-69 of SEQ ID NO:1.

[0118] In the present invention, the intracellular domain of the long-acting activation protein includes the intracellular domain of the DAP10 protein, the long-acting activation element, and an optional additional costimulatory domain.

[0119] In one embodiment, the long-acting activation protein of the present invention comprises the intracellular domain of DAP10 protein and the long-acting activation element, but does not contain an additional costimulatory domain.

[0120] In one embodiment, the sequence of the long-acting activated protein without an additional co-stimulatory domain is shown in SEQ ID NO: 3:

[0121] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSSCSGCGSLSLPLLA GLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRGGHETGRLSGA ADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 3).

[0122] In one embodiment, the long-acting activated protein of the present invention comprises the intracellular domain of the DAP10 protein, the long-acting activation element, and contains one or more additional costimulatory domains. Preferably, the additional costimulatory domain is located between the intracellular domain of the DAP10 protein and the long-acting activation element. The costimulatory domain is a costimulatory domain derived from proteins such as CD40, CD27, 4-1BB, OX40, but is not limited thereto. The multiple costimulatory domains may be costimulatory domains derived from the same protein or costimulatory domains derived from different proteins.

[0123] In a preferred embodiment, the costimulatory domain is a costimulatory domain derived from CD40 protein, and its sequence is shown in SEQ ID NO:15.

[0124] In one embodiment, the sequence of the long-acting activation protein containing a CD40 costimulatory domain is as shown in SEQ ID NO: 4:

[0125] MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRGKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK(SEQ ID NO:4)

[0126] The long-acting activated protein of the present invention can be used as a signal transduction molecule for TIL cell activation. After TIL cells recognize tumor cells, they can efficiently and persistently activate TIL cells so that they can effectively kill tumor cells. In addition, the long-acting activated protein of the present invention can also be used as an activation element for other immune cells, such as PBMC-derived T cells, NK cells, etc., but is not limited thereto.

[0127] Nucleic acid constructs of the present invention

[0128] In the present invention, a nucleic acid construct capable of expressing the long-acting activated protein of the present invention is provided. The first expression cassette of the nucleic acid construct of the present invention comprises a sequence encoding the long-acting activated protein of the present invention, and optionally comprises a hypoxia-responsive promoter.

[0129] The hypoxia-responsive promoter of the present invention is a promoter comprising n hypoxia-responsive elements HRE, i.e., n×HRE promoter. Hypoxia is a hallmark feature of solid tumors, and this unique environmental signal can be used for targeted cancer therapy, 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 expressing the fusion protein of the present invention, the present invention uses a hypoxia-responsive promoter to express the fusion protein of the present invention in an oxygen-deficient environment. There is no particular limitation on the type of promoter used to construct the hypoxia-responsive promoter of the present invention. Preferably, the present invention uses a TK mini promoter containing n×HRE.

[0130] Preferably, the present invention adopts 3×HRE-TK-mini promoter.

[0131] In one embodiment, the nucleic acid construct of the present invention further comprises a molecular switch element (also known as an immune brake element). When the cells expressing the nucleic acid construct present safety risks, the molecular switch element can be used as a target to eliminate the risky cells using corresponding drugs.

[0132] In one embodiment, the molecular switch or immune brake element of the present invention is hEGFRt. When the cells containing the nucleic acid construct of the present invention present safety risks, the hEGFRt therapeutic monoclonal antibody cetuximab can be injected to eliminate the target cells through ADCC and CDC, further improving safety; at the same time, the flow detection of hEGFRt molecules can also be used to indicate the positive rate of cells integrated with the target gene. The exemplary sequence of the molecular switch element is shown in SEQ ID NO: 16.

[0133] In the nucleic acid construct of the present invention, it is preferred that the nucleic acid construct further contains a coding sequence for expressing 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 connected to the first expression cassette via a cleavable linker peptide.

[0134] Interleukin IL-15 is a pro-survival cytokine that maintains the homeostasis of long-lived CD8+ memory T cells, inhibits activation-induced cell death (AICD), enhances anti-tumor activity in vivo, and reverses T cell anergy. Monomeric IL-15 is a small unstable protein with a short serum half-life, requiring supraphysiological administration to obtain an in vivo response. 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.

[0135] In one embodiment, the molecular switch element and the membrane-bound IL-15 element can be connected to the first expression cassette or to each other via a cleavable connecting peptide, respectively; preferably, the cleavable connecting peptide is a self-cleaving peptide; preferably, the self-cleaving peptide is selected from: T2A, P2A, or a combination thereof.

[0136] In one embodiment, the molecular switch element and the membrane-bound IL-15 element are located in separate expression cassettes and driven by separate promoters, each of which is independently a constitutive promoter or an inducible promoter.

[0137] The vector of the present invention

[0138] In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.

[0139] Some vectors are capable of autonomous replication in the host cell they introduce (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal 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, naked DNA polynucleotides, polynucleotides consisting of DNA and RNA in the same chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. that cannot autonomously replicate.

[0140] In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA techniques are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors.

[0141] 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.

[0142] Engineered TIL cells of the present invention

[0143] 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 long-acting activated protein of the present invention and / or TIL cells containing the vector of the present invention.

[0144] The engineered TIL cells expressing the long-acting activated protein of the present invention can recognize tumor cells through the NKG2D-NKG2DL pathway, reduce the escape of tumor cells, and are efficiently activated to initiate killing ability, and cell exhaustion rarely occurs, ultimately achieving efficient and lasting killing of tumor cells.

[0145] In addition, the engineered TIL cells of the present invention have excellent proliferation ability and cell viability during the culture stage, which ensures the durability of the TIL cells during the killing stage and is suitable for industrial production.

[0146] In one embodiment, the engineered TIL cells of the present invention further express the membrane-bound IL-15 of the present invention.

[0147] In one embodiment, the engineered TIL cells of the present invention further express the molecular switch element of the present invention. When the engineered TIL cells of the present invention present safety risks, the molecular switch element can be used as a target to eliminate the risky cells using corresponding drugs. The molecular switch element of the present invention is selected from the following group: hEGFRt, BCMA, CD20. Preferably, the molecular switch element of the present invention is hEGFRt.

[0148] Pharmaceutical composition of the present invention

[0149] The pharmaceutical composition of the present invention may comprise the fusion protein of the present invention or the immune effector cell of the present invention (such as the engineered TIL cell of the present invention) and one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants. These compositions may be suitable for use in the treatment of therapeutic indications described herein.

[0150] In the present invention, the term "subject" can be a mammal in need of treatment, such as a human or veterinary patient (e.g., a rodent, such as a mouse or rat, cat, dog, cow, horse, sheep, goat, or other livestock). In some embodiments, the "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like. The subject may be suspected of having a disease characterized by cell proliferation or having a disease characterized by cell proliferation, diagnosed as having a disease characterized by cell proliferation, or a control subject confirmed not to have a disease characterized by cell proliferation. As described herein, diagnostic methods for diseases characterized by cell proliferation and the clinical division of such diagnosis are known to those skilled in the art.

[0151] The pharmaceutical composition of the present invention can be used to treat tumors. In the present invention, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, neuroendocrine tumors, mesotheliomas, schwannomas, meningiomas, adenocarcinomas, and melanomas. "Tumor cells" may further include "solid tumors," which refer to tumors selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastomas, cervical cancer, ovarian cancer, bladder cancer, hepatomas, breast cancer, colon cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer.

[0152] The main advantages of the present invention include:

[0153] (1) The engineered TIL cells of the present invention can recognize and kill tumor cells through the NKG2D-NKG2DL pathway, reducing the escape of tumor cells that do not express MHC-I molecules.

[0154] (2) The engineered TIL cells of the present invention can be efficiently and durably activated after recognizing tumor cells, reducing TIL cell exhaustion and having continuous, multi-round tumor killing capabilities.

[0155] (3) The engineered TIL cells of the present invention have a high level of proliferation ability and viability during the culture stage.

[0156] (4) The engineered TIL cells of the present invention can be induced to express the long-acting activated protein of the present invention under the hypoxia environment characteristic of solid tumors through a hypoxia-responsive promoter.

[0157] (5) The engineered TIL cells of the present invention can also express immune molecule switches, such as hEGFRt, thereby avoiding the potential toxicity of continuous expression of exogenous proteins and improving the safety of clinical treatment.

[0158] 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 present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0159] Example 1: Preparation of TIL cells containing target genes

[0160] 1. Plasmid Construction

[0161] CD3ζ is a commonly used signal transduction sequence in the art, so in the present invention, CD3ζ is used as a control activation element. Figure 1 As shown, three constructs named 076, 095, and 096 were constructed, respectively, wherein: Figure 1 The simplified diagram of the plasmid structure of 076, 095, and 096 is shown; among them, 3×HRE-TK-mini Promoter is a hypoxia-inducible promoter, SP1 is an IgE signal peptide; CD86Hinge is the hinge region of CD86; CD86TM-cyto is the transmembrane region and intracellular region of D86; T2A and P2A are each self-cleaving polypeptides; hEGFRt is the third and fourth domain elements of human EGFR. The sequences of each element are shown in Table 1 below.

[0162] Table 1 Nucleotide sequence or amino acid sequence of each element

[0163]

[0164]

[0165] The molecular sequences of each construct were synthesized by GenScript, and the synthesized gene sequences were cloned into the basic vector pLV-EF1a-c-MYC-IRES-EGFP (Wuhan Miaoling) to obtain plasmids 076, 095, and 096. Transformation (Quanshijin, CD301) and plasmid extraction (Jianshi Bio, TD429) were performed using kits.

[0166] 2. Lentivirus Preparation and Titer Determination

[0167] The plasmid vectors 076, 095, 096, psPAX2 and pMD2.G obtained in the above steps were purified respectively; the plasmids were mixed and transfected with Lipo3000 in proportion to HEK-293T cells (100mm dish culture) grown to a density of 70-90% (about 80%). After 4 hours of transfection, the cells were removed, the supernatant was discarded, and 10 ml of DMEM complete medium was added to each dish; the dish was placed in a 37°C, 5% CO2 incubator for continued culture; the culture solution was collected and centrifuged for 48 hours to retain the supernatant, centrifuged at 400g for 5 minutes, and the supernatant was filtered with a 0.45μm filter. The filtrate obtained was 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.

[0168] The HEK-293T cells were washed, trypsinized and graded diluted and then counted. After infection, the HEK-293T cells were treated with hypoxia, and the proportion of cells positive for transgene expression was detected by flow cytometry to calculate the virus titer. Lentivirus titer calculation method: virus titer = (m × 2.5 × 10 5 × dilution factor) / infected volume; m is the proportion of cells positive for transgene expression.

[0169] 3. Lentiviral Infection of TIL Cells

[0170] TIL cells were isolated and cultured from tumor masses. According to the titer of lentivirus, TIL cells were infected with MOI=10; lentivirus, Lentiboost (100×), TIL cells (3.5×10 5 ) was added to REP medium, with 200 μl of medium-virus-cell mixture in each centrifuge tube. After mixing, the mixture was transferred to a 48-well plate and cultured in a 37°C, 5% CO2 incubator. After 24 hours of infection, the cells were collected and centrifuged at 1000 rpm, and the virus solution was discarded. An appropriate amount of REP (AIM V medium: A1640 medium = 1:1) complete medium was added to resuspend the mixture, and the mixture was transferred to a 24-well plate and cultured in a 37°C, 5% CO2 incubator. After 72 hours of culture, the cells were collected.

[0171] The TIL cells infected with lentivirus containing 076, 095, and 096 constructs were named 076TIL, 095TIL, and 096TIL, respectively, and the uninfected control TIL cells were named TIL-MOCK.

[0172] 4. Hypoxia treatment of TIL cells

[0173] Take 5×10 5 The cells prepared in step 3 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 (oxygen concentration of 21%).

[0174] Example 2: TIL positive rate detection

[0175] The positive rate of TIL cells in each group was analyzed by flow cytometry with indirect antibody. 5×10 5 The cells were detected by flow cytometry. Each group of cells was divided into two parts: a completely negative group and a group to be detected, with a cell volume of 50 μl in each group; IL15 antibody (Beijing Baixinyi Biotechnology Co., Ltd., A09D21-9E) was added to the group to be detected, and incubated at room temperature for 15 min; after the incubation, 1 ml of PBS containing 2% FBS was added, and the cells were centrifuged at 400 g for 5 min; 50 μl of FITC Donkey anti-rabbit IgG Antibody (BioLegend, 406403) antibody mixture prepared with PBS containing 2% FBS was added to the cells of the group to be detected, and the cells were incubated at room temperature for 10 min; 1 ml of PBS containing 2% FBS was added, and the cells were centrifuged at 400 g for 5 min; the supernatant was discarded, and 50 μl of 7-AAD mixture prepared with PBS containing 2% FBS was added to the cells of the group to be detected, and the cells were incubated at room temperature for 7 min; 1 ml of PBS containing 2% FBS was added to the completely negative group and the group to be detected, and the cells were centrifuged at 400 g for 5 min; the supernatant was discarded, and 200 μl of 2% Resuspend the cells in FBS-containing PBS and test on the instrument.

[0176] The flow cytometry results of mIL15 are shown in the figure. Figure 2 After 076, 095, and 096 lentiviruses infected TILs, IL15+ was detected by flow cytometry after 4 days of culture. The flow cytometry data showed that the positive rates of 076, 095, and 096 were 44.4%, 44.0%, and 41.5%, respectively, all of which had high levels of positive infection rates.

[0177] Example 3: TIL proliferation ability and viability detection

[0178] After lentivirus infection of TIL, the ratio of 1:25 with trophoblast cells (IL-21NK cell expansion reagent, Zhongying Biological Product No.: ZY-NKZ-0104) was adjusted to a cell density of 5×10 5 Cells / ml were cultured in REP medium in 6-well plates, 2 ml / well. Cell counts were performed every two days and fresh medium was added to adjust the cell density to 5×10 5 The cells were cultured continuously for 9 days, and the cell count and viability were determined. Each group of experiments was repeated three times.

[0179] The proliferation folds of TIL-MOCK, 076TIL, 095TIL and 096TIL are as follows Figure 3 As shown, they are 255, 150, 245, and 260 times, respectively. The proliferation times of 095TIL, 096TIL, and control TIL are basically the same, while the proliferation times of 076TIL is significantly reduced.

[0180] After 9 days of expansion and culture, the cell viability of TIL-MOCK, 076TIL, 095TIL and 096TIL was as follows: Figure 4 As shown, they are 93%, 85%, 94%, and 92% respectively. The cell viabilities of 095TIL, 096TIL, and control TIL are comparable, while the cell viability of 076TIL is reduced.

[0181] The above results show that for each group of TIL cell proliferation times and cell viability, the 076TIL cells containing the control activation element were lower than the control group, while the 095TIL and 096TIL cells containing the long-acting activation element were equivalent to the control TIL, indicating that the long-acting activation element is more conducive to the proliferation of engineered TIL cells.

[0182] Example 4: Detection of TIL IFN-γ secretion

[0183] The secretion of IFN-γ after co-culture of TIL cells and tumor cells in each group was detected. TIL: tumor cells = 5:1 were co-cultured with HELA (highly expressing NKG2DL) for 18 hours, and the supernatant was collected and tested with an IFN-γ ELISA kit. The experiment was performed using the Human IFN-γ Precoated ELISA Kit (Dakoway, 2307-3), and the operation was performed according to the instructions of the kit, as follows:

[0184] Add the diluted Cytokine standard to the standard well, 100μl / well, dilute the sample with Dilution bufferR (1×), add the sample to the sample well, 100μl / well. Cover with a sealing film and incubate at room temperature for 2 hours. Remove the liquid in the well and add 1×Washing buffer working solution, 300μl / well; leave for 1 minute and discard the liquid in the well. Repeat 3 times, and dry on filter paper each time. Add Biotinylated antibody working solution, 100μl / well. Cover with a sealing film and incubate at room temperature for 1 hour. Add Streptavidin-HRP working solution, 100μl / well. Cover with a sealing film and incubate at room temperature (18-25℃) for 30 minutes. Add TMB, 100μl / well, incubate at room temperature, away from light for 5-30 minutes, and determine the termination of the reaction based on the depth of the color in the well (dark blue). Usually 10-20 minutes of color development can achieve good results. Stop solution was added immediately, 100 μl / well, to terminate the reaction. OD values ​​were read at 450 nm using a microplate reader. Data were analyzed using the four-parameter method.

[0185] ELISA test results Figure 5 As shown, the secretion of IFN-γ by 076TIL, 095TIL, and 096TIL after co-culture with various tumor cells was much higher than that of uninfected TIL-MOCK.

[0186] The above results show that 076TIL, 095TIL, and 096TIL all have significant IFN-γ secretion compared with the control cells, indicating that the control activation element and the long-acting activation element of the present invention can activate TIL cells using the NKG2D-NKG2L recognition pathway.

[0187] Example 5: Continuous killing ability of TIL on tumor cells

[0188] The ability of various TILs to continuously kill tumor cells was tested. The experimental schematic diagram is shown below. Figure 6 The specific operation is as follows: take 1×10 5 HeLa-COGFP tumor cells (stable expression of COGFP) were seeded in 24-well plates. After 24 hours, TIL cells were diluted to a density of 1.5×10 6 / ml. Aspirate the culture medium in the original wells of the 24-well plate. Add the prepared TILs of each group at a ratio of TIL:tumor cells = 5:1. After every 24 hours, aspirate 50% of the culture (including TIL) in the wells and add 1×10 5The 24-well plate with tumor cells HELA-COGFP was co-cultured for 24 hours, and the above experiment was repeated three times. The remaining tumor cells in the wells were washed with PBS each time, and the activity of tumor cells was detected by CCK8 (Mei Lun Bio, MA0218-5), and the continuous killing of tumor cells by TIL was determined.

[0189] Microscope observation results Figure 7 As shown: The HELA cell control group showed that the HELA cells were in good growth state, and the cells grew in a dense monolayer. When HELA was co-cultured with 076TIL, 095TIL and 096TIL for the first time, the HELA cells grew slowly and failed to fill the cell culture wells. Most of the cells became round and fell off, indicating that 076TIL, 095TIL and 096TIL all had a strong killing effect on tumor cells HELA;

[0190] In the second co-culture experiment, HELA cells in the HELA+095TIL and 096TIL experimental groups became round and fell off in a large area, showing obvious TIL killing of tumor cells, while only a small number of HELA cells fell off in the 076TIL plus HELA experimental group, indicating that the killing ability of 076TIL gradually weakened, while the killing ability of 095TIL and 096TIL did not change significantly compared with the first round;

[0191] In the third co-culture experiment, HELA cells in the HELA+095TIL and 096TIL experimental groups still showed large-scale rounding and shedding, indicating obvious TIL killing of tumor cells, while in the 076TIL plus HELA experimental group, almost no obvious HELA cell shedding was observed, indicating that the killing ability of 076TIL on HELA cells was significantly weakened, while the killing ability of 095TIL and 096TIL remained at a high level.

[0192] The results of detecting tumor cell activity by CCK8 method are as follows Figure 8 As shown: Compared with 076TIL, 095TIL and 096TIL have higher continuous killing ability against tumor cells. After three rounds of killing, the cytotoxicity of 076TIL has been significantly reduced, while 095TIL and 096TIL maintained high cytotoxicity in the three rounds of killing, indicating that 095TIL and 096TIL both have excellent ability to continuously kill tumor cells, among which the continuous killing ability of 095TIL is slightly better than that of 096TIL.

[0193] The above results indicate that the engineered TIL cells comprising the long-acting activated protein of the present invention have a high level and lasting tumor cell killing ability.

[0194] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An engineered TIL cell, characterized in that: The engineered TIL cells express an exogenous long-acting activated protein, wherein the long-acting activated protein comprises a DAP10 element and a long-acting activated element, and the sequence of the long-acting activated element is shown in SEQ ID NO: 2; Wherein, the long-acting activated protein has a structure as shown in Formula I: L-ECD-TM-ICD1-ICD2-ICD3 (Formula I) In the formula, "-" are each independently a peptide bond or a connecting peptide; L is none or signal peptide; ECD is the extracellular domain, the sequence of which is shown at positions 19-48 of SEQ ID NO: 1; TM is a transmembrane domain, the sequence of which is shown at positions 49-69 of SEQ ID NO: 1; ICD1 is the intracellular domain from the DAP10 protein, and its sequence is shown at positions 70-92 of SEQ ID NO: 1; ICD2 has no or a costimulatory domain, the sequence of the costimulatory domain is shown in SEQ ID NO: 15; ICD3 is a long-acting activation element.

2. The engineered TIL cell according to claim 1, characterized in that The ICD2 is a co-stimulatory domain from the CD40 protein.

3. The engineered TIL cell according to claim 1, characterized in that The amino acid sequence of the long-acting activated protein is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

4. The engineered TIL cell according to claim 1, characterized in that The engineered TIL cells contain a polynucleotide encoding the long-acting activated protein.

5. The engineered TIL cell according to claim 1, characterized in that The engineered TIL cells contain a vector, which includes: a polynucleotide encoding the long-acting activation protein.

6. The engineered TIL cell according to claim 1, characterized in that The engineered TIL cells also express a membrane-bound IL-15 fusion polypeptide comprising the following elements fused together: (i) interleukin 15 (IL-15); (ii) CD86 transmembrane domain and CD86 intracellular domain.

7. The engineered TIL cell according to claim 1, characterized in that The engineered TIL cells contain a nucleic acid construct, which includes a polynucleotide encoding the long-acting activated protein.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the engineered TIL cells as described in claim 1, and a pharmaceutically acceptable carrier.

9. A kit, characterized in that: The kit comprises the engineered TIL cells according to claim 1, or a reagent for preparing the engineered TIL cells according to claim 1, wherein the reagent is selected from the following group: (Y1) a polynucleotide encoding the long-acting activated protein; or (Y2) A vector comprising: a polynucleotide encoding the long-acting activated protein.

10. Use of the engineered TIL cells in the preparation of a drug according to claim 1, characterized in that: The medicine is used for preventing, alleviating and / or treating tumors.

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