Engineered til cell with enhanced sustained killing capacity and use thereof

Abstract

Provided are an engineered TIL cell with an enhanced sustained killing capacity and the use thereof. Specifically, provided is an engineered TIL cell expressing a long-acting activation element, wherein the surface thereof contains a modified DAP10 fusion protein, which can effectively initiate the activation of the TIL cell while reducing the exhaustion caused by the overactivation of TIL. The engineered TIL cell can recognize a tumor cell by means of an NKG2D-NKG2DL pathway, so as to reduce an escape of the tumor cell. In addition, in a culture stage, the engineered cell of the engineered TIL cell has an excellent expansion capacity and cell viability. The engineered TIL cell can efficiently and persistently kill tumor cells, and has application prospects in the field of tumor treatment.

Description

Engineered tils with enhanced continuous killing ability and applications thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to engineered TIL cells with enhanced continuous killing ability and applications thereof. BACKGROUND

[0002] Adoptive cellular immunotherapy (ACI or AIT) refers to transferring immune cells with anti-tumor activity to tumor patients, especially genetically engineered immune cells, to achieve direct killing of tumor cells or killing of tumor cells by stimulating the body's immune response. The currently widely used cellular immunotherapy includes CAR-T, TCR-T, CAR-NK, etc. TIL cells, i.e. tumor infiltrating lymphocytes, play an important role in the specific immune response of the body to tumor cells, and have their outstanding advantages compared with other immune cells.

[0003] Firstly, tumor infiltrating lymphocytes TIL can recognize multiple tumor-specific neoantigens and tumor-associated antigens, making them more effective in dealing with tumor heterogeneity; secondly, TIL usually contains a certain number of effector memory T cells, which express chemokine receptors after being stimulated by tumor antigens in vivo, making them more easily localized to tumor tissues after reinfusion; in addition, TIL is derived from the patient himself, and has lower toxicity. Therefore, TIL therapy has shown great potential in the field of solid tumors.

[0004] However, TIL cells derived from tumor tissue are often in a terminal differentiation and exhaustion state after activation, resulting in weakened killing ability of TIL cells to tumor cells; most of the TIL cells cultured in vitro are non-tumor-specific bystander cells, and how to convert bystander cells into effector cells with recognition and killing function to tumor cells to improve the therapeutic effect of TIL cells is one of the problems to be solved in the field of genetically engineered TILs. In addition, tumor cells often escape the killing of immune cells by reducing the expression of MHC-I molecules or TAP gene mutation, etc. to lose antigen presentation function.

[0005] Therefore, there is an urgent need in the art to develop engineered TIL cells with high and persistent tumor cell killing ability. SUMMARY

[0006] The purpose of the present application is to provide engineered TIL cells with high and persistent tumor cell killing ability.

[0007] In a first aspect of the present application, there is provided an engineered TIL cell expressing an exogenous long-acting activating protein comprising a DAP10 element and a long-acting activating element, the sequence of which is shown as SEQ ID NO: 2.

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

[0009] (1) an extracellular domain comprising a DAP10 element or an active fragment thereof;

[0010] (2) a transmembrane domain; and

[0011] (3) an intracellular signaling domain comprising a DAP10 intracellular domain and a long-acting activating element.

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

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

[0014] wherein,

[0015] each of “-” is independently a peptide bond or a linking peptide;

[0016] L is nothing or a signal peptide;

[0017] ECD is an extracellular domain;

[0018] TM is a transmembrane domain

[0019] ICD1 is an intracellular domain from a DAP10 protein;

[0020] ICD2 is nothing or a costimulatory domain;

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

[0022] In another preferred embodiment, the L is nothing.

[0023] In another preferred embodiment, the L is a signal peptide selected from the group consisting of DAP10, GM-CSF receptor alpha, CD8, CD28.

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

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

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

[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 set forth in SEQ ID NO: 1 at positions 49-69.

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

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

[0032] In another preferred embodiment, the ICD2 is none.

[0033] In another preferred embodiment, the ICD2 is one, two, or more costimulatory 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 costimulatory domain from a CD40 protein.

[0035] In another preferred embodiment, the sequence of the ICD2 is set forth in SEQ ID NO: 15.

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

[0037] In another preferred embodiment, the amino acid sequence of the long-acting activating protein is set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0038] In another preferred embodiment, the engineered TIL cell has the long-acting activating protein on the cell membrane thereof.

[0039] In another preferred embodiment, the engineered TIL cell has one or more properties selected from the group consisting of:

[0040] (1) a persistent tumor cell killing ability;

[0041] (2) a reduced exhaustion of TIL cells;

[0042] (3) a reduced immune escape and recurrence of a tumor;

[0043] (4) improved in vivo expansion capacity.

[0044] In another preferred embodiment, the activation is self-activation, i.e., the engineered TIL cells expressing the long-lasting activating protein are activated.

[0045] In another preferred embodiment, 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-lasting activating 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 comprising a polynucleotide encoding the long-lasting activating protein.

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

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

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

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

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

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

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

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

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

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

[0059] In another preferred embodiment, the amino acid sequence of the CD86 transmembrane domain and the intracellular domain is set forth in SEQ ID NO: 10.

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

[0061] In another preferred embodiment, the engineered TIL cell comprises a nucleic acid construct comprising a polynucleotide encoding the long-acting activator protein.

[0062] In another preferred embodiment, the nucleic acid construct comprises a promoter selected from the group consisting of 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 number of hypoxia response elements (HREs), i.e., an n x HREs-containing promoter, and n is an integer selected from 1-20.

[0065] In another preferred embodiment, n is an integer selected from 2-9; more preferably, n is an integer selected from 2-4; most preferably, n is 3.

[0066] In another preferred embodiment, the hypoxia-responsive promoter is selected from the group consisting of an n x HREs-containing TK-mini promoter, an n x HREs-containing CMV-mini promoter, and an n x HREs-containing IL2-mini promoter.

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

[0068] In another preferred embodiment, 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 selected from the group consisting of 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 activator 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 linker peptide-encoding sequence.

[0072] In another preferred embodiment, the cleavable linker 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 one expression cassette.

[0074] In a second aspect of the present application, there is provided a pharmaceutical composition comprising the engineered TIL cell of the first aspect of the present application, and a pharmaceutically acceptable carrier.

[0075] In a third aspect of the present application, there is provided a kit comprising the engineered TIL cell of the first aspect of the present application, or reagents for preparing the engineered TIL cell of the first aspect of the present application, wherein the reagents are selected from the group consisting of:

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

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

[0080] 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 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, biliary tumor, and head and neck cancer.

[0081] In a fifth aspect of the present application, there is provided a method of treating a disease, the method comprising administering the cell of the first aspect of the present application and / or the pharmaceutical composition of the second aspect of the present application 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 a cancer.

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

[0085] 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, hepatoma, 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 application, each of the technical features described above and each of the technical features described in detail below (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0087] Figure 1 shows a schematic diagram of the construction of 076, 095, and 096 molecules.

[0088] Figure 2 shows the results of detecting the infection positive rate of TIL cells in each group.

[0089] Figure 3 shows the results of detecting the proliferation ability of TIL cells in each group.

[0090] Figure 4 shows the results of detecting the cell viability of TIL cells in each group after 9-day expansion culture.

[0091] Figure 5 shows the results of detecting the secretion amount of cytokine IFN-γ of TIL cells in each group co-cultured with tumor cells.

[0092] Figure 6 shows a schematic diagram of the experimental design for detecting the continuous killing ability of TIL cells on tumor cells.

[0093] Figure 7 shows the microscope observation of the continuous killing results of TIL cells in each group on tumor cells.

[0094] Figure 8 shows the results of detecting the cytotoxicity of TIL cells in each group during the continuous killing process of tumor cells. DETAILED DESCRIPTION

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

[0096] Activation of TIL cells

[0097] Natural TIL cells are activated by specific recognition of tumor cells through MHC-I molecules on the surface of tumor cells, and have the ability to specifically kill tumor cells. However, most of the TIL cells obtained by in vitro culture are non-tumor-specific bystander cells, which cannot be well activated. In addition, tumor cells often escape the killing of TIL cells by reducing the expression of MHC-I molecules and other ways. In the present application, TIL cells are engineered so that TIL cells can be activated through the NKG2D-NKG2DL pathway, improving 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 and CD8+ T cells, which plays an important role in innate immunity and participates in the killing of tumor cells by various immune cells. NKG2D ligand (NKG2DL) is not expressed in normal cells, but is highly expressed on the surface of various tumor cells of different sources (such as colorectal cancer, liver cancer, brain glioma, etc.). NK, NKT cells can directly recognize and bind each other through NKG2D-NKG2DL form, thereby killing tumor cells.

[0099] However, in TIL cells, the downstream molecule DAP10 of NKG2D only plays a costimulatory signal role, and TIL cells cannot effectively kill tumor cells after recognizing tumor cells through NKG2D-NKG2DL due to the lack of the first activating signal. Therefore, in the present application, TIL cells are engineered to be able to recognize and kill tumor cells through the NKG2D-NKG2DL pathway.

[0100] TIL cells derived from tumor tissue are often in a terminal differentiation and exhaustion state after activation, which limits the continuous killing ability of TIL cells on tumor cells. In addition, the existing engineered TIL cells are prone to be in an over-activated state, and obvious exhaustion phenomenon also occurs during the killing process, which is not conducive to the persistent and effective killing of tumor cells by TIL cells.

[0101] In the present application, long-acting activated engineered TIL cells expressing the long-acting activated protein of the present application are provided, which have excellent continuous tumor cell killing function.

[0102] DAP10

[0103] DAP10 exerts its unique intracellular signaling function after binding with cell receptors such as NKG2D, is highly conserved in the evolution process, and the sequences of DAP10 of mammals (especially primates) are highly homologous and have similar functions. The homology of DAP10 of humans and non-human primates is as high as 90-100%, and the homology of DAP10 of humans and rodents is about 80%.

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

[0105] Among them, the signal peptide of DAP10 is 1-18 of SEQ ID NO: 1, the extracellular domain is 19-48, the transmembrane domain is 49-69, and the intracellular domain is 70-92.

[0106] In the present application, 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%) of the function of wild-type DAP10. It should be understood that in the present application, DAP10 also includes mutant proteins whose functions are greater than wild-type DAP10, for example, mutant DAP10 with ≥100% (such as 100-200%) of the function of wild-type DAP10. In the present application, the mutation of DAP10 can be naturally occurring or artificially introduced.

[0107] Long-acting activating protein of the present application

[0108] As used herein, the term "long-acting activating protein of the present application" refers to a fusion protein containing a DAP10 element and a long-acting activating element of the present application, which has a TIL cell activating function.

[0109] In the present application, the "long-acting activating element" is a sequence with signal transmission function screened, and its amino acid sequence is shown as SEQ ID NO: 2:

[0110] In the present application, the long-acting activating element also includes a sequence having sequence identity with SEQ ID NO: 2, for example, a sequence having ≥85% (for example, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity with SEQ ID NO: 2, and having the same or similar TIL cell activating function as the sequence shown in SEQ ID NO: 2.

[0111] The long-acting activating protein of the present application is a membrane-bound fusion protein comprising a DAP10 element and a long-acting activating element, specifically comprising an extracellular domain, a transmembrane domain, an intracellular domain of DAP10 and a long-acting activating element, and optionally comprising a signal peptide, and optionally comprising one or more additional co-stimulatory domains.

[0112] In one embodiment, the long-acting activating protein of the present application contains a signal peptide. The signal peptide is a segment of polypeptide located at the N of the fusion protein, which plays a guiding role in the process of protein folding, transportation and localization. In the present application, the selection of the signal peptide is not particularly limited, including the signal peptides commonly used in the art, such as: DAP10 signal peptide, GM-CSF receptor alpha 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 the sequence thereof is shown in SEQ ID NO: 1, positions 1-18. In one embodiment, the sequence of the signal peptide is shown in SEQ ID NO: 12.

[0113] In one embodiment, the long-acting activating protein of the present application does not contain a signal peptide. In the process of protein maturation, the signal peptide is generally removed, so that the long-acting activating protein which has been localized on the cell membrane does not contain a signal peptide.

[0114] The extracellular domain of the long-acting activating protein of the present application is an extracellular domain from DAP10 protein, preferably human DAP10. In one embodiment, the sequence of the extracellular domain is shown in SEQ ID NO: 1, positions 19-48.

[0115] In the present application, the transmembrane domain of the long-acting activating protein is not particularly limited, preferably a transmembrane domain from DAP10, CD8, or CD28, more preferably a transmembrane domain from DAP10. In one embodiment, the sequence of the transmembrane domain is shown in SEQ ID NO: 1, positions 49-69.

[0116] In the present application, the intracellular domain of the long-acting activating protein comprises an intracellular domain of DAP10 protein, a long-acting activating element, and optionally an additional co-stimulatory domain.

[0117] In one embodiment, the long-acting activating protein of the present application comprises an intracellular domain of DAP10 protein, a long-acting activating element, and does not contain an additional co-stimulatory domain.

[0118] In one embodiment, the long-acting activating protein without an additional co-stimulatory domain has a sequence shown in SEQ ID NO: 3:

[0119] In one embodiment, the long-acting activating protein of the present application comprises the intracellular domain of DAP10 protein, a long-acting activating element, and contains one or more additional co-stimulatory domains. Preferably, the additional co-stimulatory domain is located between the intracellular domain of DAP10 protein and the long-acting activating element. The co-stimulatory domain is derived from the co-stimulatory domain of CD40, CD27, 4-1BB, OX40, etc., but is not limited thereto. The multiple co-stimulatory domains can be co-stimulatory domains derived from the same protein or co-stimulatory domains derived from different proteins.

[0120] In a preferred embodiment, the co-stimulatory domain is derived from the co-stimulatory domain of CD40 protein, the sequence of which is shown in SEQ ID NO: 15.

[0121] In one embodiment, the long-acting activating protein containing the CD40 co-stimulatory domain has the sequence shown in SEQ ID NO: 4:

[0122] The long-acting activating protein of the present application can serve as a signal transduction molecule for the activation of TIL cells, and after the TIL cells recognize tumor cells, the long-acting activating protein can efficiently and durably activate the TIL cells, enabling the TIL cells to effectively kill tumor cells. In addition, the long-acting activating protein of the present application can also serve as an activating element for other immune cells, such as T cells, NK cells, etc. derived from PBMC, but is not limited thereto.

[0123] Nucleic acid construct of the present application

[0124] In the present application, a nucleic acid construct capable of expressing the long-acting activating protein of the present application is provided. The first expression cassette of the nucleic acid construct of the present application comprises a sequence encoding the long-acting activating protein of the present application, and optionally comprises a hypoxia-responsive promoter.

[0125] The hypoxia-responsive promoter of the present application is a promoter comprising n hypoxia-responsive elements HRE, i.e., an n x HRE promoter. Hypoxia is a hallmark feature of solid tumors, and this unique environmental signal can be used for cancer-targeted therapy, enabling the target gene to be highly expressed in the tumor microenvironment and not expressed or lowly expressed in normal tissues, thereby maximizing the function of the exogenous gene while minimizing possible side effects. In the present application, a hypoxia-responsive promoter is used when constructing a nucleic acid molecule expressing the fusion protein of the present application, so as to express the fusion protein of the present application in a hypoxic environment. The type of promoter used to construct the hypoxia-responsive promoter of the present application is not particularly limited, and preferably, the TK mini promoter containing n x HRE is used in the present application.

[0126] Preferably, the 3 x HRE-TK-mini promoter is used in the present application.

[0127] In one embodiment, the nucleic acid construct of the present application further comprises a molecular switch element (also known as an immune brake element). When the cells expressing the nucleic acid construct pose a safety risk, the molecular switch element can be used as a target to eliminate the risky cells using corresponding drugs.

[0128] In one embodiment, the molecular switch or immune brake element of the present application is hEGFRt. When the cells containing the nucleic acid construct of the present application pose a safety risk, the therapeutic monoclonal antibody cetuximab can be injected to eliminate the target cells through ADCC and CDC effects, further improving the safety; at the same time, the flow cytometry detection of the hEGFRt molecule can be used to indicate the positive rate of the cells integrated with the target gene. The exemplary sequence of the molecular switch element is shown in SEQ ID NO: 16.

[0129] In the nucleic acid construct of the present application, preferably, it further comprises a coding sequence of an expression of a 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 through a cleavable linker peptide.

[0130] Interleukin IL-15 is a pro-survival cytokine that can maintain long-lived CD8+ memory T cell homeostasis, suppress activation-induced cell death (AICD), enhance anti-tumor activity in vivo and reverse T cell anergy. Monomeric IL-15 is a small unstable protein with a short serum half-life, and requires supraphysiological dosing to achieve an in vivo response. By connecting IL-15 with a transmembrane domain and an intracellular domain, a membrane-bound IL-15 is obtained, which has the function of maintaining the long-term persistence of the memory stem cell phenotype. Preferably, the intracellular domain is the intracellular domain of CD86, and preferably the transmembrane domain is the transmembrane domain of CD86.

[0131] 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 through a cleavable linker peptide, respectively; preferably, the cleavable linker peptide is a self-cleaving peptide; preferably, the self-cleaving peptide is selected from: T2A, P2A, or a combination thereof.

[0132] In one embodiment, the molecular switch element and the membrane-bound IL-15 element are located in independent expression cassettes and are driven by independent promoters. The promoters are each independently a constitutive promoter or an inducible promoter.

[0133] Vector of the present application

[0134] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been 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, wherein additional DNA segments can be ligated into the viral genome.

[0135] Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Examples of non-episomal mammalian vectors include naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides consisting of DNA and RNA in the same strand, poly-lysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, etc. which are not capable of autonomous replication.

[0136] 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 of utility in recombinant DNA technologies are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0137] As used herein, "vector of the present application" refers to a vector containing a nucleic acid construct of the present application. Preferably, the vector of the present application is a plasmid.

[0138] Engineered TIL cells of the present application

[0139] As used herein, the terms "engineered TIL cells of the present application" or "TIL cells of the present application" are used interchangeably and refer to TIL cells capable of expressing a long-acting activating protein of the present application and / or TIL cells containing a vector of the present application.

[0140] Engineered TIL cells expressing a long-acting activating protein of the present application can recognize tumor cells through the NKG2D-NKG2DL pathway, reduce the escape of tumor cells, and be efficiently activated to initiate killing ability, with little occurrence of cell exhaustion, ultimately achieving efficient and persistent killing of tumor cells.

[0141] In addition, engineered TIL cells of the present application have excellent expansion capacity and cell viability during the culture stage, ensuring the persistence of TIL cells during the killing stage, and are suitable for industrial production.

[0142] In one embodiment, the engineered TIL cells of the present application further express a membrane-bound IL-15 of the present application.

[0143] In one embodiment, the engineered TIL cells of the application further express a molecular switch element of the application. The molecular switch element can serve as a target for elimination of the risky cells with the corresponding drug when the engineered TIL cells of the application present a safety risk. The molecular switch element of the application is selected from the group consisting of hEGFRt, BCMA, CD20. Preferably, the molecular switch element of the application is hEGFRt.

[0144] Pharmaceutical compositions of the application

[0145] The pharmaceutical compositions of the application can comprise a fusion protein of the application or an immune effector cell of the application (such as an engineered TIL cell of the application) and one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants. These compositions can be suitable for use in therapy of the therapeutic indications described herein.

[0146] In the present application, the term "subject" can be a mammal in need of treatment, such as a human or a veterinary patient (e.g., a rodent, such as a mouse or rat, a cat, a dog, a cow, a horse, a sheep, a goat, or other livestock). In some embodiments, a "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like. The subject can be suspected of having a disease characterized by cell proliferation or having a disease characterized by cell proliferation, diagnosed as having a disease characterized by cell proliferation, or a control subject confirmed not to have a disease characterized by cell proliferation, as described herein, diagnostic methods for a disease characterized by cell proliferation and clinical partitioning of such diagnosis are known to one skilled in the art.

[0147] The pharmaceutical compositions of the application can be used to treat a tumor. In the present application, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, neuroendocrine tumors, mesotheliomas, schwannomas, meningiomas, adenocarcinomas, and melanomas. "Tumor cells" can further include "solid tumors," which refers to a tumor selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary duct tumor, and head and neck cancer.

[0148] The main advantages of the present application include:

[0149] (1) The engineered TIL cells of the present application can recognize and kill tumor cells through the NKG2D-NKG2DL pathway, reducing the escape of tumor cells that do not express MHC class I molecules.

[0150] (2) The engineered TIL cells of the present application can be activated efficiently and durably after recognizing tumor cells, reducing the exhaustion of TIL cells, and having continuous and multiple rounds of tumor killing ability.

[0151] (3) The engineered TIL cells of the present application have high proliferation ability and viability during the culture stage.

[0152] (4) The engineered TIL cells of the present application can induce expression of the long-acting activated protein of the present application under the hypoxic environment characteristic of solid tumors through the hypoxia-responsive promoter.

[0153] (5) The engineered TIL cells of the present application can also express an immune molecule switch such as hEGFRt, avoiding the potential toxicity of continuous expression of exogenous proteins and improving the safety of clinical treatment.

[0154] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0155] Example 1: Preparation of TIL cells containing target genes

[0156] 1. Plasmid construction

[0157] CD3ζ is a commonly used signal transduction sequence in the art, and therefore is used as a control activation element in the present application. As shown in FIG. 1, three constructs named 076, 095, and 096 were constructed, respectively. FIG. 1A shows the simple structure of the 076, 095, and 096 plasmids. In the figure, 3xHRE-TK-mini Promoter is a hypoxia-inducible promoter, SP1 is an IgE signal peptide, CD86Hinge is a CD86 hinge region, CD86TM-cyto is the transmembrane region and intracellular region of D86, T2A and P2A are self-cleavage polypeptides, respectively, and hEGFRt is the third and fourth domain element of human EGFR. The sequences of each element are shown in Table 1 below.

[0158] Table 1: Nucleotide sequences or amino acid sequences of each element

[0159] The molecular sequence of each construct was synthesized by Kingsway Biotech, and the synthesized gene sequence was cloned into the basic vector pLV-EF1a-c-MYC-IRES-EGFP (Wuhan Moli) to obtain 076, 095, and 096 plasmids. The kit was used for transformation (Kangshi Biological, CD301) and plasmid extraction (Jianshi Biological, TD429).

[0160] 2. Lentivirus preparation and titer determination

[0161] The 076, 095, and 096 plasmid vectors obtained in the above steps, psPAX2 vector, and pMD2.G vector were purified and large-scale purified, respectively; the plasmids were mixed and transfected into HEK-293T cells (100 mm dish culture) at a proportion of Lipo3000 to 70-90% (about 80%) density. 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; it was placed in a 37°C, 5% CO2 incubator for continuous culture; after 48 h of culture, the culture solution was centrifuged to retain the supernatant, and the supernatant was filtered with a 0.45 μm filter to obtain the original solution of the recombinant lentivirus; the lentivirus was concentrated using an Utra-15 centrifugal filter device at 5000 rpm for 50 min.

[0162] The HEK-293T cells after washing, trypsin digestion, and gradient dilution were counted. After infection, the HEK-293T cells were subjected to hypoxic treatment, and the proportion of cells positive for transgene expression was detected by flow cytometry to calculate the virus titer. The lentivirus titer calculation method: virus titer = (m x 2.5 x 10 5 x dilution factor) / volume of infection; m is the proportion of cells positive for transgene expression.

[0163] 3. Lentivirus infection of TIL cells

[0164] The TIL cells were isolated and cultured from tumor blocks. The TIL cells were infected at MOI = 10 according to the titer of the lentivirus; the lentivirus, Lentiboost (100x), and TIL cells (3.5 x 10 5 ) were added to REP medium, and each centrifuge tube contained 200 μl of medium-virus-cell mixture, which was mixed and transferred to a 48-well plate for culture in a 37°C, 5% CO2 incubator; after 24 h of infection, the cells were collected and centrifuged at 1000 rpm, and the virus solution was discarded; an appropriate amount of REP (AIM V medium:A1640 medium = 1:1) complete medium was added for resuspension, and the cells were transferred to a 24-well plate for culture in a 37°C, 5% CO2 incubator; after 72 h of culture, the cells were collected.

[0165] The TIL cells infected by the lentivirus containing 076, 095, and 096 constructs are named 076TIL, 095TIL, and 096TIL, respectively, and the uninfected control TIL cells are named TIL-MOCK.

[0166] 4. Hypoxic treatment of TIL cells

[0167] 5x10 5 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 hypoxic treatment (experimental group); the remaining TIL cells (as a non-hypoxic control group) were transferred to a new 24-well plate and placed in a 37°C, 5% CO2 incubator (oxygen concentration of 21%) for culture.

[0168] Example 2: Detection of TIL positive rate

[0169] The positive rates of TIL cells in each group were analyzed by intercalator antibody flow cytometry analysis. 5x10 5 cells in the experimental and control groups were subjected to flow cytometry analysis. Each group of cells was divided into two parts: a full negative group and a detection group, and each group of cells had a volume of 50 μl; IL15 antibody (Beijing Bainexin Biotechnology Co., Ltd., A09D21-9E) was added to the detection group, and the mixture was incubated at room temperature for 15 min; after incubation, 1 ml of PBS containing 2% FBS was added, and centrifugation was performed at 400g for 5 min; 50 μl of FITC Donkey anti-rabbit IgG Antibody (BioLegend, 406403) antibody mixture prepared using PBS containing 2% FBS was added to the detection group cells, and incubation was performed at room temperature for 10 min; 1 ml of PBS containing 2% FBS was added, and centrifugation was performed at 400g for 5 min; the supernatant was discarded, and 50 μl of 7-AAD mixture prepared using PBS containing 2% FBS was added to the detection group cells, and incubation was performed at room temperature for 7 min; 1 ml of PBS containing 2% FBS was added to the full negative and detection groups, and centrifugation was performed at 400g for 5 min; the supernatant was discarded, and 200 μl of PBS containing 2% FBS was added to resuspend the cells, and the cells were subjected to detection.

[0170] The results of flow cytometry detection of mIL15 are shown in FIG. 2. After TIL was infected with 076, 095, and 096 lentivirus, respectively, and cultured for 4 days, IL15+ was detected by flow cytometry. According to the flow cytometry data, the positive rates of 076, 095, and 096 were 44.4%, 44.0%, and 41.5%, respectively, all with high levels of positive infection rate.

[0171] Example 3: Detection of TIL proliferation ability and viability

[0172] After lentivirus infection of TIL, the TIL was co-cultured with the trophoblast cells (IL-21 NK cell expansion reagent, ZY Biosciences, ZY-NKZ-0104) at a ratio of 1:25, and the cell density was adjusted to 5 x 105cells / ml, and the cells were cultured in REP medium in a 6-well plate at 2 ml / well. Cell counting was performed every two days, and the cell density was adjusted to 5 x 105cells / ml by adding fresh medium. The cells were continuously cultured for 9 days, and cell counting and viability determination were performed, with three replicates in each group. 5 5 The cell density was adjusted to 5 x 105cells / ml by adding fresh medium. The cells were continuously cultured for 9 days, and cell counting and viability determination were performed, with three replicates in each group.

[0173] The proliferation fold of TIL-MOCK, 076TIL, 095TIL, and 096TIL is shown in Figure 3, which is 255, 150, 245, and 260, respectively. The proliferation fold of 095TIL, 096TIL, and the control TIL is basically the same, and the proliferation fold of 076TIL is significantly reduced.

[0174] The cell viability of TIL-MOCK, 076TIL, 095TIL, and 096TIL after 9 days of expansion culture is shown in Figure 4, which is 93%, 85%, 94%, and 92%, respectively. The cell viability of 095TIL, 096TIL, and the control TIL is comparable, and the cell viability of 076TIL is reduced.

[0175] The above results show that the proliferation fold and cell viability of each group of TIL cells are lower than the control group for 076TIL cells containing the control activation element, while the 095TIL and 096TIL cells containing the long-acting activation element are comparable to the control TIL, indicating that the long-acting activation element is more conducive to the proliferation of engineered TIL cells.

[0176] Example 4: Detection of TIL IFN-γ secretion

[0177] The amount of IFN-γ secreted by each group of TIL cells after co-culture with tumor cells was detected. TIL: tumor cells = 5:1 were co-cultured with HELA (high expression of NKG2DL) for 18 hours, and the supernatant was collected for detection using an IFN-γ ELISA detection kit. The Human IFN-γ Precoated ELISA Kit (Dakow, 2307-3) kit was used for the experiment, and the operation was performed according to the kit instructions, as follows:

[0178] ​Add diluted Cytokine standard to standard wells, 100 μl / well. Dilute samples with Dilution buffer R (1x) and add to sample wells, 100 μl / well. Cover the plate with sealing film and incubate at room temperature for 2 hours. Remove the liquid from the wells, add 1x Washing buffer working solution, 300 μl / well; leave for 1 minute and discard the liquid from the wells. Repeat 3 times, each time blotting dry on filter paper. Add Biotinylated antibody working solution, 100 μl / well. Cover the plate with sealing film and incubate at room temperature for 1 hour. Add Streptavidin-HRP working solution, 100 μl / well. Cover the plate with sealing film and incubate at room temperature (18-25°C) for 30 minutes. Add TMB, 100 μl / well, and incubate at room temperature, protected from light, for 5-30 minutes. Stop the reaction according to the intensity of the color in the wells (dark blue). Usually 10-20 minutes of color development gives good results. Stop the reaction by adding Stop solution, 100 μl / well. Read the OD values at 450 nm in a microplate reader. Analyze the data using the 4-parameter method.

[0179] The results of ELISA detection are shown in Figure 5. 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.

[0180] The above results show that 076TIL, 095TIL and 096TIL all have significant secretion of IFN-γ compared with the control cells, indicating that the control activation element and the long-acting activation element of the application can activate TIL cells using the NKG2D-NKG2L recognition pathway.

[0181] Example 5: Continuous killing ability of TIL on tumor cells

[0182] The continuous killing ability of various TIL on tumor cells was detected, and the experimental schematic diagram is shown in Figure 6. The specific operation is as follows: 1x10 5 HLELA-COGFP (stably expressing COGFP) tumor cells were inoculated in a 24-well plate. After 24 hours, the TIL cells were diluted to a density of 1.5x10 6 / ml. The culture solution in the original well of the 24-well plate was aspirated. The prepared TIL of each group was added according to TIL:tumor cells = 5:1. After every 24 hours, 50% of the culture containing TIL was removed from the well and 1x10 5The 24-well plate of tumor cells HELA-COGFP was continuously co-cultured for 24 hours, and the above experiment was repeated continuously for 3 times. The remaining tumor cells in the wells were washed with PBS each time, and the tumor cell activity was detected by CCK8 (Milen Biotech, MA0218-5) method to perform continuous killing of tumor cells by TIL.

[0183] The microscopic observation results are shown in Figure 7: the HELA cell control group shows that the HELA cells grow well, and the cells grow in a dense monolayer. When the HELA cells are co-cultured with 076TIL, 095TIL and 096TIL for the first time, the HELA cells grow slowly, and most of the cells are round and fall off, which indicates that 076TIL, 095TIL and 096TIL all have strong killing effect on tumor cells HELA;

[0184] In the second co-culture experiment, the HELA cells in the HELA+095TIL and 096TIL experimental groups are round and fall off in a large area, and obvious killing of tumor cells by TIL occurs, while only a small part of the HELA cells fall off in the 076TIL+HELA experimental group, which indicates that the killing ability of 076TIL gradually weakens, while the killing ability of 095TIL and 096TIL does not change significantly compared with the first round;

[0185] In the third co-culture experiment, the HELA cells in the HELA+095TIL and 096TIL experimental groups are still round and fall off in a large area, and obvious killing of tumor cells by TIL occurs, while almost no obvious fall-off of the HELA cells can be seen in the 076TIL+HELA experimental group, which indicates that the killing ability of 076TIL on the HELA cells significantly weakens, while the killing ability of 095TIL and 096TIL remains at a high level.

[0186] The results of CCK8 method for detecting tumor cell activity are shown in Figure 8: compared with 076TIL, 095TIL and 096TIL have higher continuous killing ability on tumor cells, and after three rounds of killing, the cytotoxicity of 076TIL has been significantly reduced, while 095TIL and 096TIL both maintain high cytotoxicity in the three rounds of killing, which indicates that 095TIL and 096TIL both have excellent continuous killing ability on tumor cells, and the continuous killing ability of 095TIL is slightly better than that of 096TIL.

[0187] The above results show that the engineered TIL cells containing the long-acting activated protein of the application have high and persistent tumor cell killing ability.

[0188] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. An engineered TIL cell, characterized in that, The engineered TIL cell expresses an exogenous long-acting activating protein comprising a DAP10 element and a long-acting activating element, the sequence of which is shown in SEQ ID NO:

2.

2. The engineered TIL cell of claim 1, wherein, The long-acting activating protein comprises, from N-terminus to C-terminus, the following elements fused together: (1) an extracellular domain comprising a DAP10 element or an active fragment thereof; (2) a transmembrane domain; and (3) an intracellular signaling domain comprising a DAP10 intracellular domain and a long-acting activating element.

3. The engineered TIL cell of claim 1, wherein, The long-acting activating protein has a structure as shown in Formula I: L-ECD-TM-ICD1-ICD2-ICD3 (Formula I) In the formula, each of “-” is independently a peptide bond or a connecting peptide; L is nothing or a signal peptide; ECD is an extracellular domain; TM is a transmembrane domain ICD1 is an intracellular domain from a DAP10 protein; ICD2 is nothing or a costimulatory domain; ICD3 is a long-acting activating element.

4. The engineered TIL cell of claim 3, wherein The ECD is an extracellular domain from a DAP10 protein.

5. The engineered TIL cell of claim 3, wherein The ICD2 is one, two or more costimulatory domains derived from a protein selected from the group consisting of CD40, CD27, 4-1BB, OX40, or a combination thereof.

6. The engineered TIL cell of claim 1, wherein The amino acid sequence of the long-acting activating protein is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

7. The engineered TIL cell of claim 1, wherein The engineered TIL cell comprises a polynucleotide encoding the long-acting activating protein.

8. The engineered TIL cell of claim 1, wherein, The engineered TIL cell comprises a vector comprising a polynucleotide encoding the long-acting activating protein.

9. The engineered TIL cell of claim 1, wherein The engineered TIL cell further expresses a membrane-bound IL-15 fusion polypeptide comprising the following elements fused together: (i) interleukin 15 (IL-15); (ii) a CD86 transmembrane domain and a CD86 intracellular domain.

10. The engineered TIL cell of claim 1, wherein The engineered TIL cell comprises a nucleic acid construct comprising a polynucleotide encoding the long-acting activating protein.

11. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the engineered TIL cell of claim 1, and a pharmaceutically acceptable carrier.

12. A kit characterized in that, The kit comprises the engineered TIL cell of claim 1, or reagents for preparing the engineered TIL cell of claim 1, wherein the reagents are selected from the group consisting of: (Y1) a polynucleotide encoding the long-acting activating protein; or (Y2) a vector comprising a polynucleotide encoding the long-acting activating protein.

13. Use of the engineered TIL cells of claim 1 in the manufacture of a medicament, wherein, The medicament is used for preventing, alleviating and / or treating a tumor.

14. The method of claim 13, wherein, The tumor comprises a solid tumor, a hematological tumor, or a combination thereof.

15. A method for treating a disease, characterized in that, The method comprises administering the engineered TIL cell of claim 1 and / or the pharmaceutical composition of claim 11 to a subject in need.

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