CN117511885A - Engineered TIL cell capable of improving tumor recognition and killing capability and application thereof - Google Patents

Abstract

The invention provides an engineering TIL cell capable of improving the capability of recognizing and killing tumors and application thereof. The invention relates to the technical field of genetic engineering and cell therapy. The engineered TIL cell surface of the invention contains exogenous membrane-bound DAP10-CD3 zeta fusion protein, and the endoplasmic reticulum or golgi apparatus contains retention type fusion protein. Specifically, the invention provides a membrane-bound DAP10-CD3 zeta fusion protein, which enables TIL cells to be activated by the activated fusion protein after recognizing tumor cells through an NKG2D-NKG2DL path, thereby effectively killing the tumor cells. The invention also provides a retention type fusion protein, so that endogenous NKG2DL expressed when TIL cells are activated can be combined with the retention type fusion protein, thereby being retained in cells, avoiding mutual identification and killing among the TIL cells, and improving the activity rate of the TIL cells.

Description

Engineered TIL cell capable of improving tumor recognition and killing capability and application thereof

Technical Field

The invention relates to the technical field of genetic engineering and cell therapy, in particular to an engineering TIL cell capable of improving the capability of recognizing and killing tumors and application thereof.

Background

Tumor-infiltrating lymphocyte (TIL) cell therapy refers to therapy in which tumor-infiltrating lymphocytes are isolated from tumor tissue, cultured in vitro and expanded in large quantities, and then returned to the patient. TIL is composed of T cells with multiple TCR clones, and can recognize multiple tumor-specific neoantigens and tumor-associated antigens, making them more effective against tumor heterogeneity. TIL usually contains effector memory T cells that express chemokine receptors in vivo after stimulation with tumor antigens, making them more easily localized to tumor tissue after reinfusion. In addition, TIL is derived from the patient himself, with low toxicity. TIL therapy has shown great potential in the field of solid tumors.

The TIL cells of the tumor-derived tissue are enriched for tumor-specific T cells, however, these tumor-specific T cells are often in a terminally differentiated, depleted state after tumor-specific activation, resulting in a reduced killing capacity of the TIL cells against the tumor cells. In addition, the problem of how to transform bystander cells into effector cells with recognition and killing functions on tumor cells is urgently solved in the cultured TIL cells, wherein most of the cells are bystander cells with non-tumor specificity. In addition, tumor cells often lose antigen presenting functions due to reduced expression of MHC-I molecules, mutation of TAP gene, or the like, thereby escaping killing of immune cells.

The NKG2D molecules on the surface of TIL cells can bind to NKG2DL molecules on the surface of tumor cells, thereby allowing TIL cells to recognize tumor cells. However, TIL cells transiently express NKG2DL in an activated state, which causes mutual killing between TIL cells, resulting in a significant decrease in the viability of TIL cells.

Thus, there is a strong need in the art to develop engineered TIL cells that recognize and kill tumor cells, reduce immune escape, and reduce TIL cell self-killing.

Disclosure of Invention

The invention aims to provide an engineering TIL cell which has the functions of identifying and killing tumor cells, reducing immune escape and reducing self-phase killing of the TIL cell and application thereof.

In a first aspect of the invention, there is provided an engineered tumor-infiltrating lymphocyte (TIL cell) that expresses an exogenous fusion protein selected from the group consisting of: an activated fusion protein, a retentive fusion protein, or a combination thereof;

wherein the activated fusion protein is a membrane-bound DAP10-CD3 zeta fusion protein;

the retention-type fusion protein comprises: NKG2D extracellular domain and KDEL sequences.

In another preferred embodiment, the engineered TIL cells express both an activated fusion protein and a resident fusion protein.

In another preferred embodiment, the activated fusion protein comprises: (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 cd3ζ;

wherein the extracellular domain and intracellular signaling domain are connected in series by the transmembrane domain.

In another preferred embodiment, the engineered TIL cells have the activated fusion protein on their cell membranes; and/or said engineered TIL cells have said retention-type fusion protein in the endoplasmic reticulum or golgi apparatus.

In another preferred embodiment, the engineered TIL cell contains a polynucleotide encoding the activated fusion protein and/or the retentate fusion protein.

In another preferred embodiment, the engineered TIL cell contains a nucleic acid construct comprising a first expression cassette comprising a polynucleotide encoding the activated fusion protein and/or a polynucleotide encoding the resident fusion protein.

In another preferred embodiment, the engineered TIL cells comprise a vector comprising: polynucleotides encoding the activated fusion protein and/or polynucleotides encoding the retentate fusion protein.

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

(i) Interleukin 15 or a functionally active fragment thereof;

(ii) A transmembrane domain; and

(iii) An intracellular domain;

wherein the intracellular domain is a CD86 intracellular domain.

In another preferred embodiment, the CD86 intracellular domain comprises a full-length intracellular domain, or an active fragment thereof.

In another preferred embodiment, the IL-15 comprises full-length, mature forms of IL-15, or active fragments thereof.

In another preferred embodiment, the IL-15 includes wild-type and mutant forms.

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

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

In another preferred embodiment, the fusion polypeptide further optionally comprises a signal peptide and/or linker (linker).

In another preferred embodiment, the linker is located between the element (i) and the element (ii), between the element (ii) and the element (iii), between the element (i) and the hinge region, or between the hinge region and the element (ii).

In another preferred embodiment, the connector comprises a flexible connector or a rigid connector.

In another preferred embodiment, the amino acid sequence of the linker is shown as SEQ ID NO. 7.

In another preferred embodiment, the fusion polypeptide has the structural formula (I):

X-IL15-L-H-TM-Cyto (I)

wherein,

"-" are each independently absent or linked peptides;

x is none or a signal peptide;

IL15 is an interleukin 15 element;

l is none or a linker;

h is a hinge region;

TM is the transmembrane domain;

cyto is an intracellular domain, wherein the intracellular domain is a CD86 intracellular domain.

In another preferred embodiment, the transmembrane domain is a CD86 transmembrane domain.

In another preferred embodiment, the amino acid sequences of the CD86 transmembrane and intracellular domains are shown in SEQ ID NO. 9.

In another preferred embodiment, the hinge region is a CD86 hinge region, preferably the amino acid sequence of the CD86 hinge region is shown in SEQ ID NO. 7.

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

In another preferred embodiment, the engineered TIL cells have one or more properties selected from the group consisting of:

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

(2) Reducing the ability of activated TIL cells to recognize and kill each other;

(3) Expanded TIL cells recognize the target antigen profile;

(4) Can effectively prevent tumor immune escape and recurrence.

In a second aspect of the invention, there is provided an activated fusion protein comprising:

(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 cd3ζ;

wherein the extracellular domain and intracellular signaling domain are connected in series by the transmembrane domain.

In another preferred embodiment, the intracellular domain of DAP10 is linked to CD3 zeta either directly or via a linking peptide. (preferably, the length of the connecting peptide is 1-10aa, preferably 1-6aa, more preferably 1-3 aa).

In another preferred embodiment, the intracellular signaling domain further comprises a co-stimulatory domain.

In another preferred embodiment, the intracellular signaling domain further comprises one, two or more co-stimulatory domains.

In another preferred embodiment, the co-stimulatory domain is a co-stimulatory domain from a protein selected from the group consisting of: CD28, CD27, 4-1BB, CD40, OX40, or combinations thereof.

In another preferred embodiment, the co-stimulatory domain is located between the intracellular domain of DAP10 and CD3 zeta and/or is located at the C-terminus of CD3 zeta.

In another preferred embodiment, where the intracellular signaling domain comprises an additional costimulatory domain, the DAP10 intracellular domain and the costimulatory domain, and/or the cd3ζ and the costimulatory domain, may be linked directly or via a linking peptide; preferably, the length of the linker peptide is 1-10aa, more preferably 1-6aa, most preferably 1-3aa.

In another preferred embodiment, the fusion protein further comprises a signal peptide at the N-terminus.

In another preferred embodiment, the signal peptide is selected from the group consisting of: DAP10 signal peptide, CD8a signal peptide, CD28 signal peptide.

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

In another preferred embodiment, the amino acid sequence of the extracellular domain is shown at positions 19-48 of SEQ ID NO. 1.

In another preferred embodiment, the transmembrane domain is a transmembrane domain from DAP10, CD8a, or CD 28.

In another preferred embodiment, the amino acid sequence of the transmembrane domain is shown at positions 49-69 of SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the intracellular domain of DAP10 in the intracellular signal domain is shown at positions 70-92 of SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of CD3 zeta in the intracellular signal domain is shown in positions 93-206 of SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the intracellular signaling domain is shown at positions 70-206 of SEQ ID NO. 1.

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

In another preferred embodiment, the fusion polypeptide has one or more properties selected from the group consisting of:

(a) The fusion protein has the function of combining with NKG 2D;

(b) Activating T cells (TIL cells);

(c) Activating NK cells.

In another preferred embodiment, the activation is self-activation (i.e., immune cells expressing the membrane-bound DAP10-CD3 zeta fusion protein are activated).

In another preferred embodiment, the activated immune cells (T cells, NK cells) can effectively kill target cells by the interaction of NKG2D-NKG2 DLs.

In a third aspect of the invention, there is provided a retentive fusion protein comprising: NKG2D extracellular domain and KDEL sequences.

In another preferred embodiment, said KDEL sequence of said retention type fusion protein is located C-terminal to the extracellular domain of NKG 2D.

In another preferred embodiment, the NKG2D extracellular domain of the retention-type fusion protein is shown in positions 22-156 of SEQ ID NO. 2.

In another preferred embodiment, the NKG2D extracellular domain and KDEL sequence are linked directly or via a linker peptide; preferably, the length of the linker peptide is 1-15aa, more preferably 1-10aa, most preferably 1-5aa.

In another preferred embodiment, the KDEL sequence is located at positions 157-160 of SEQ ID NO. 2.

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

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

In another preferred embodiment, the retention type fusion protein binds to endogenous NKG2DL, thereby retaining endogenous NKG2DL in the cell.

In a fourth aspect of the invention there is provided a polynucleotide encoding an activated fusion protein according to the second aspect of the invention and/or a retentate fusion protein according to the third aspect of the invention.

In another preferred embodiment, the polynucleotide encodes an activated fusion protein according to the second aspect of the invention.

In another preferred embodiment, the polynucleotide encodes a retentive fusion protein of the third aspect of the invention.

In another preferred embodiment, the polynucleotide encodes an activated fusion protein according to the second aspect of the invention and a retentate fusion protein according to the third aspect of the invention.

In another preferred embodiment, the activated fusion protein and the retentate fusion protein are linked by a cleavable linker peptide.

In another preferred embodiment, the cleavable linking peptide is a self-cleaving peptide; preferred self-cleaving peptides are selected from the group consisting of: T2A peptide, P2A peptide, or a combination thereof.

In a fifth aspect of the invention there is provided a nucleic acid construct comprising a first expression cassette comprising a polynucleotide of the fourth aspect of the invention.

In another preferred embodiment, the first expression cassette further comprises a promoter.

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

In another preferred embodiment, the promoter is selected from the group consisting of: constitutive promoters, inducible promoters, or combinations thereof.

In another preferred embodiment, the promoter comprises a hypoxia responsive promoter.

In another preferred embodiment, the hypoxia responsive promoter is a promoter comprising n hypoxia responsive elements HREs, i.e.a promoter comprising n X HRE elements, n being an integer selected from 1 to 20.

In another preferred embodiment, n is an integer selected from 2 to 9; more preferably, n is an integer selected from 2 to 4; optimally, n is 3.

In another preferred embodiment, the hypoxia responsive promoter is selected from the group consisting of: TK-mini promoter containing n X HRE element, CMV-mini promoter containing n X HRE element, IL2-mini promoter containing n X HRE element.

In another preferred embodiment, the hypoxia responsive promoter is a 3 XHRE TK-mini promoter.

In another preferred embodiment, the nucleic acid construct further comprises a sequence of molecular switching elements selected from the group consisting of: hEGFRt, BCMA, CD20.

In another preferred embodiment, the molecular switching element is hEGFRt.

In another preferred embodiment, the molecular switching element is linked to the first expression cassette by a cleavable linker peptide sequence.

In another preferred embodiment, the cleavable linking peptide is a self-cleaving 2A peptide; preferably a T2A peptide.

In another preferred embodiment, the nucleic acid construct comprises a second expression cassette comprising a second promoter and a molecular switch element coding sequence.

In another preferred embodiment, the second promoter is an SFFV promoter.

In a sixth aspect of the invention there is provided a vector comprising a polynucleotide according to the fourth aspect of the invention.

In another preferred embodiment, the vector comprises a plasmid or a viral vector.

In another preferred embodiment, the viral vector comprises: lentiviral vectors, adenoviral vectors, yellow fever viral vectors.

In another preferred embodiment, the vector is a plasmid.

In a seventh aspect of the invention, there is provided a composition comprising an engineered TIL cell according to the first aspect of the invention, an activated fusion protein according to the second aspect of the invention, a retention fusion protein according to the third aspect of the invention, a polynucleotide according to the fourth aspect of the invention, a nucleic acid construct according to the fifth aspect of the invention, and/or a vector according to the sixth aspect of the invention, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the composition comprises the engineered TIL cells of the first aspect of the invention and a pharmaceutically acceptable carrier.

In an eighth aspect of the invention, there is provided a kit comprising an engineered TIL cell according to the first aspect of the invention, an activated fusion protein according to the second aspect of the invention, a retentate fusion protein according to the third aspect of the invention, a polynucleotide according to the fourth aspect of the invention, a nucleic acid construct according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, and/or a composition according to the seventh aspect of the invention.

In another preferred embodiment, the kit comprises the engineered TIL cells according to the first aspect of the invention, or an agent for preparing the engineered TIL cells according to the first aspect of the invention, wherein the agent is selected from the group consisting of:

(Y1) a polynucleotide encoding said activated fusion protein and said retentate fusion protein; or (b)

(Y2) a carrier comprising: polynucleotides encoding the activated fusion protein and/or polynucleotides encoding the retentate fusion protein.

In a ninth aspect of the invention there is provided the use of an engineered TIL cell according to the first aspect of the invention, an activated fusion protein according to the second aspect of the invention, a retentate fusion protein according to the third aspect of the invention, a polynucleotide according to the fourth aspect of the invention, a nucleic acid construct according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a composition according to the seventh aspect of the invention, and/or a kit according to the eighth aspect of the invention in the preparation of a medicament for the prevention, alleviation and/or treatment of a tumour.

In another preferred embodiment, there is provided the use of an engineered TIL cell according to the invention, or of a kit according to the invention, for the preparation of a medicament for the treatment of a tumor.

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, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

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

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

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

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, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a schematic representation of an engineered TIL killer tumor cell of the present invention.

FIG. 2 shows a schematic structural diagram of the 055, 073, 076 and 083 constructs of the present invention.

FIG. 3 shows the results of a detection of TIL positive rate by flow detection of lentiviral infection, where TIL mock is the control TIL. The abscissa represents the relative fluorescence intensity, and the trapezoid represents the proportion of positive cells.

FIG. 4 shows the results of the assay for the proliferative capacity of TIL cells in each group, where TIL-MOCK is the control TIL.

FIG. 5 shows the results of detection of TIL cell viability in each group, wherein TIL-MOCK is a control TIL.

FIG. 6 shows the results of stem cell duty cycle detection for each group of TIL cells, where TIL-MOCK is the control TIL. The abscissa represents relative fluorescence intensity, and the Q2 region represents the proportion of CD45RA and CD62L biscationic cells, indicating the proportion of stem T cells.

FIG. 7 shows the results of IFN-gamma secretion assays for each group of TIL cells, where Ctr MOCK is control TIL.

FIG. 8 shows a photograph of each group of TIL cells co-cultured with tumor cells (HELA) at a ratio of 3:1 for 24 hours and then observed microscopically, wherein TIL-MOCK is a control TIL.

FIG. 9 shows the results of cell killing experiments of tumor cells (HELA) by each group of TIL cells, wherein HELA only is tumor cells only and TIL-MOCK is control TIL.

FIG. 10 shows the results of cell killing experiments of tumor cells (A-375B 2M KO) by TIL cells of each group.

Fig. 11 shows the effect of the molecular switch of the present invention. The abscissa represents the relative fluorescence intensity, and the (q2+q3) region represents the proportion of apoptotic cells.

Detailed Description

As a result of extensive and intensive studies, the present inventors have for the first time provided an engineered TIL cell whose cell surface contains an exogenous membrane-bound DAP10-CD3 zeta fusion protein, and whose endoplasmic reticulum or Golgi apparatus contains a retention type fusion protein. The membrane-bound DAP10-CD3 zeta fusion protein (activated fusion protein) is expressed in TIL cells, and can unexpectedly and effectively start the activation of the TIL cells, thereby more effectively killing tumor cells. Specifically, after the engineered TIL cells of the present invention recognize NKG2DL on the surface of tumor cells through NKG2D on the surface thereof, the TIL cells can be further activated effectively by the fusion protein of the present invention, thereby allowing the TIL cells to kill tumor cells effectively. Experiments show that the retention type fusion protein can be effectively retained by an endoplasmic reticulum, so that NKG2DL endogenously expressed by TIL cells is retained in the cells, the mutual killing among the TIL cells is avoided, the brittleness in the TIL cell culture process is prevented, and the activity of the TIL cells is obviously improved. The engineered TIL cells of the invention are useful for treating tumors. The present invention has been completed on the basis of this finding.

DAP10

DAP10 is widely expressed in immune cells, is highly conserved during evolution, and exerts its unique intracellular signaling function through binding to cell receptors such as NKG 2D. DAP10 conducts cell activation signals through its YINM motif, providing costimulatory signals in T cells.

The amino acid sequence accession number of the human DAP10 is GenBank AAD46986.1, 92 amino acids are shown in positions 1-92 of SEQ ID NO. 1. DAP consists of multiple parts of a signal peptide (positions 1-18), an extracellular domain (positions 19-48), a transmembrane domain (positions 49-69), and an intracellular domain (positions 70-92), wherein the intracellular domain comprises a YINM motif.

The sequence of DAP10 in mammals, especially primates, is highly homologous and functionally similar. Human and non-human primate DAP10 homologies up to 90-100%, human and rodent DAP10 homologies at about 80%.

In the present invention, DAP10 includes both wild-type and mutant DAP10, so long as the mutant DAP10 retains or substantially retains the function of the wild-type DAP10, such as retaining ≡50% (preferably ≡60%,. Gtoreq.70%,. Gtoreq.80%,. Gtoreq.90%, and even ≡100% (e.g., 100-200%)) of the function of the wild-type DAP10. It is to be understood that DAP10 in the present invention also includes mutant proteins that function more than wild-type DAP10, such as mutant DAP10 having ≡100% (e.g., 100-200%) of the function of wild-type DAP10.

In the present invention, mutations of DAP10 can be naturally occurring or artificially introduced.

NKG2D and NKG2DL

NKG2D is an activating receptor expressed on the surface of NK cells, NKT and cd8+ T cells, plays an important role in innate immunity, and is involved in killing tumor cells by various immune cells. NKG2D ligand (NKG 2 DL) family proteins, mainly comprising six cytomegalovirus UL16 binding proteins 1-6 and MHC I chain-related molecules a and B (MICA, MICB). NKG2DL is not substantially expressed in normal cells, and has high levels of expression on the cell surface of various tumors of different origins (including but not limited to colorectal cancer, liver cancer, glioma, etc.).

NKG2D is a DAP 10-associated receptor, and the receptor complex is a hexamer consisting of one NKG2D homodimer and two DAP10 homodimers.

After NKG2D binds to tumor cell surface NKG2DL, charged amino acid residues of NKG2D homodimer transmembrane region are linked to TM residues of DAP10 via two salt bridges to form a hexamer structure, which in turn induces phosphorylation of yin m motifs in the cytoplasm, which then activates downstream phosphoinositide 3 kinase (PI 3K) signaling pathways, delivering an activation signal. The signals generated by NKG2D can directly activate NK cells to exert a killing effect, and act as co-stimulatory signals to promote activation of and enhance killing effects of αβ T cells and γδ T cells.

NK and NKG cells can kill tumor cells through NKG2D-NKG2DL binding, and CD8+ T cells can recognize and kill tumors which do not express MHC-I molecules through NKG2D-NKG2 DL.

However, TIL cells themselves highly express NKG2D molecules, but since DAP10, a downstream molecule of NKG2D, only plays a role in co-stimulatory signaling in T cells, TIL cells cannot kill tumor cells efficiently due to lack of the first signaling for T cell activation after recognizing tumor cells by NKG2D-NKG2 DL.

In the present invention, an engineered TIL cell capable of being activated by the NKG2D-NKG2DL pathway to kill tumor cells is provided.

DAP10 and CD3 zeta fusion proteins of the invention

As used herein, the terms "activated fusion protein of the invention", "membrane-bound DAP10-cd3ζ fusion protein of the invention" and "DAP 10 and cd3ζ fusion protein of the invention" are used interchangeably to refer to DAP10 and cd3ζ fusion proteins of the invention having T cell activating functions. The membrane-bound DAP10-CD3 zeta fusion proteins of the invention have an extracellular structure derived from DAP10, a transmembrane domain, an intracellular domain, and an intracellular structure derived from CD3, such as CD3 zeta.

In the membrane-bound DAP10-CD3 zeta fusion proteins of the invention, the transmembrane region may be the transmembrane region from DAP10, the transmembrane region of CD3, or the transmembrane region from other membrane proteins, or a synthetic transmembrane region.

In addition, in the membrane bound DAP10-CD3 zeta fusion proteins of the invention, the hinge region or additional linking region may or may not be present between the extracellular structure and the transmembrane region, and the additional linking region may or may not be present between the intracellular structure (e.g., CD3 zeta) and the transmembrane region.

In a preferred embodiment, the extracellular structure and transmembrane region of the membrane-bound DAP10-CD3 zeta fusion proteins of the invention are derived from the DAP10 protein, preferably from the human DAP10 protein, such as from amino acids 1-69 of the human DAP10 protein.

Typically, the activated fusion proteins of the invention comprise the following elements:

(1) DAP10 element or an active fragment thereof; and

(2) An intracellular signaling domain derived from the DAP10 intracellular segment and cd3ζ.

In another preferred embodiment, the membrane-bound DAP10-CD3 zeta fusion proteins of the invention comprise (1) an extracellular, transmembrane, and intracellular domain derived from DAP10 element or an active fragment thereof, in tandem from N-terminus to C-terminus; and (2) an intracellular signaling domain derived from cd3ζ.

In one embodiment, the intracellular signaling domain optionally further comprises a co-stimulatory molecule; the co-stimulatory molecule is selected from the group consisting of: CD28, CD27, 4-1BB, CD40, OX40.

The amino acid sequence of a preferred activated fusion protein of the invention is as follows:

MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQDGKVYINMPGRGMRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1)；

Wherein the sequence of the signal peptide is shown as 1 st-18 th positions in SEQ ID NO. 1, the sequence of the extracellular domain is shown as 19 th-48 th positions in SEQ ID NO. 1, the sequence of the transmembrane domain is shown as 49 th-69 th positions in SEQ ID NO. 1, the sequence of the intracellular domain of DAP10 is shown as 70 th-92 th positions in SEQ ID NO. 1, and the sequence of CD3 zeta is shown as 93 rd-206 th positions in SEQ ID NO. 1.

The membrane-bound DAP10-CD3 zeta fusion protein can be used as a signal for TIL cell activation. Specifically, after the NKG2D of the TIL cells is combined with the NKG2DL on the surface of the tumor cells, the TIL cells can be successfully activated by the membrane-bound DAP10-CD3 zeta fusion protein of the invention, so that the TIL cells can be killed.

The inventive retention type fusion protein (tNKG 2D-KDEL)

The NKG2D ligand (NKG 2 DL) is not only highly expressed on the surface of tumor cells, but also transiently expressed on activated TIL cells, so that the activated TIL cells can recognize and kill each other.

In the present invention, there is provided a structurally unique retention type fusion protein (abbreviated as "retention type fusion protein of the present invention") comprising an NKG2D extracellular domain element and a KDEL element connected in series.

The retention type fusion protein of the present invention can retain endogenous NKG2DL produced by T cells (such as TIL cells) in cells, and reduce NKG2DL appearing on the surface of TIL cells, even eliminate NKG2DL appearing on the surface of TIL cells.

In the present invention, KDEL is Lys-Asp-Glu-Leu (also referred to as KDEL signal sequence), usually located at the carboxy-terminus (C-terminus) of the protein. The KDEL sequence has corresponding receptors on the membrane of the Golgi apparatus, and once it enters the Golgi apparatus, it is bound by the receptors on the Golgi apparatus, and forms reflux vesicles which are transported back to the endoplasmic reticulum, and the KDEL sequence is called an endoplasmic reticulum retention sequence.

Preferably, the present invention provides a retentive fusion protein expressed in TIL cells, which fuses the extracellular domain of NKG2D with a KDEL signal sequence to form a tNKG2D-KDEL protein.

After TIL cell activation, transiently expressed endogenous NKG2DL will specifically bind to the NKG2D extracellular domain in the inventive retention-type fusion protein and thus be retained in the endoplasmic reticulum, thereby avoiding or eliminating the occurrence of expressed endogenous NKG2DL on the TIL cell membrane. Therefore, mutual recognition and killing among TIL cells can be effectively prevented, and cell fragility is reduced, so that the TIL cell activity rate is improved.

An amino acid sequence of a representative retention type fusion protein of the invention is as follows:

MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVKDEL (SEQ ID NO: 2)；

wherein, the 22 nd bit to 156 th bit in SEQ ID NO. 2 are NKG2D extracellular region sequences; the 157 th to 160 th positions are KDEL sequences.

Preferably, the retentive fusion proteins of the present invention also contain a signal peptide sequence. A preferred signal peptide is the CD8a signal peptide, which has the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 3).

The nucleic acid construct of the invention

As used herein, the nucleic acid constructs of the invention may express the fusion proteins and/or the retention-type fusion proteins of the invention. The nucleic acid constructs of the invention comprise sequences encoding the fusion proteins and/or the retention type fusion proteins of the invention, and optionally include a hypoxia responsive promoter.

The hypoxia responsive promoter of the invention is a promoter comprising n hypoxia responsive elements HREs, i.e. an n×hre promoter. Hypoxia is a characteristic feature of solid tumors, and the unique environmental signal can be used for cancer targeted therapy, so that the target gene is highly expressed in the tumor microenvironment but not expressed or lowly expressed in normal tissues, the function of the exogenous gene is exerted to the maximum extent, and the possible side effects are reduced. In constructing nucleic acid molecules expressing the fusion proteins of the present invention, hypoxia responsive promoters are employed to express the fusion proteins of the present invention in a hypoxic environment. The kind of promoter used to construct the hypoxia responsive promoter of the present invention is not particularly limited, and preferably, the present invention employs a TK mini promoter containing an nxHRE.

Preferably, the 3 XHRE-TK-mini promoter is used in the present invention.

In one embodiment, the nucleic acid construct of the invention further comprises a molecular switching element (also known as an immune braking element). When the cells of the expressed nucleic acid construct present a safety risk, the molecular switching element can be used as a target, and the at-risk cells are cleared with the corresponding drug.

In one embodiment, the molecular switching element of the invention may be linked to the first expression cassette by a cleavable linker peptide; preferably, the cleavable linking peptide is a self-cleaving peptide; preferably, the self-cleaving peptide is selected from: T2A, P a, or a combination thereof.

In one embodiment, the molecular switching element of the invention is located in a second expression cassette, the expression of which is controlled by a second promoter. In one embodiment, the second promoter is an SFFV promoter, which is a constitutive promoter that stably and at high levels initiates expression of the molecular switching element. In one embodiment, the second expression cassette comprises a second signal peptide.

In one embodiment, the molecular switch or immunobraking element of the present invention is hEGFRt. When the safety risk of the cells containing the nucleic acid construct of the invention appears, the hEGFRt therapeutic monoclonal antibody cetuximab can be injected, and the target cells are cleared through ADCC and CDC actions, so that the safety is further improved; meanwhile, the flow detection of hEGFRt molecules can be utilized to indicate the cell positive rate of target gene integration.

Membrane-bound IL-15

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

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

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

The membrane-bound IL-15 may further comprise a hinge region, a signal peptide and/or a linker. Preferably, the membrane-bound IL-15 has a structural formula shown in formula (I):

X-IL-15-L-H-TM-Cyto (I)

Wherein,

"-" are each independently absent or linked peptides;

x is none or a signal peptide;

IL-15 is interleukin 15;

l is none or a linker;

h is a hinge region; the preferred hinge region is a CD86 hinge region;

TM is the transmembrane domain; the preferred transmembrane domain is the CD86 transmembrane domain;

cyto is an intracellular domain, and a preferred intracellular domain is the CD86 intracellular domain.

The vector of the invention

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 other DNA segments may be ligated. Another class of vectors are viral vectors, wherein other DNA segments may be ligated into the viral genome. 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, thereby replicating with the host genome, such as naked RNA polynucleotides that are not autonomously replicable, naked DNA polynucleotides, polynucleotides that consist of DNA and RNA in the same strand, poly-lysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, and the like. 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 technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector.

As used herein, "vector of the invention" refers to a vector containing a nucleic acid construct of the invention. Preferably, the vector of the invention is a plasmid.

Engineered TIL cells of the invention

As used herein, the term "engineered TIL cells of the invention" or "TIL cells of the invention" are used interchangeably, and refer to TIL cells capable of expressing the fusion proteins of the invention and/or TIL cells containing the vectors of the invention. A schematic representation of TIL cell recognition and killing of tumor cells of the present invention is shown in FIG. 1.

The engineered TIL cells of the invention express DAP10 and cd3ζ fusion proteins of the invention as activation signals. After the NKG2D of the engineering TIL cell is combined with the NKG2DL on the surface of the tumor cell, the NKG2D can be successfully activated by the fusion protein of DAP10 and CD3 zeta so as to kill the tumor cell.

In one embodiment, the engineered TIL cells of the invention further express a retention-type fusion protein in which the extracellular region of NKG2D of the invention is fused with a KDEL sequence, and endogenous NKG2DL transiently expressed after surface activation of the TIL cells specifically binds to the NKG2D region in the retention-type fusion protein, thereby being retained in the endoplasmic reticulum, but not on the TIL cell membrane, preventing mutual recognition and killing between TIL cells, reducing the occurrence of cell fragility, and increasing the cell viability.

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

In one embodiment, the engineered TIL cells of the invention further express the molecular switching elements of the invention. When the engineered TIL cells of the invention present a safety risk, the molecular switch element can be used as a target, and the risky cells are cleared by the corresponding drugs. The molecular switching element of the present invention is selected from the group consisting of: hEGFRt, BCMA, CD20. Preferably, the molecular switching element of the present invention is hEGFRt.

Pharmaceutical compositions of the invention

The pharmaceutical compositions of the invention may comprise the fusion proteins of the invention or the immune effector cells of the invention (e.g., the engineered TIL cells of the invention) together with one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants. These compositions may be suitable for use in the treatment of the therapeutic indications described herein.

The composition may be a liquid solution, suspension, emulsion, sustained release formulation or powder, and may be formulated with a pharmaceutically acceptable carrier. The composition may be formulated as a suppository using conventional binders and carriers such as triglycerides. By "pharmaceutically acceptable carrier" is meant a carrier matrix or vehicle (vehicle) that does not interfere with the effectiveness of the biological activity of the active ingredient and does not produce toxicity to the host or subject.

The fusion protein or immune effector cell may be delivered with a pharmaceutically acceptable vehicle. In one embodiment, the vehicle may enhance stability and/or delivery properties. Vehicles such as artificial membrane vesicles (including liposomes, nonionic surfactant vesicles (noisomes), nanolipid vesicles, etc.), microparticles or microcapsules, or colloidal formulations comprising pharmaceutically acceptable polymers.

Pharmaceutical compositions comprising one or more fusion proteins or immune effector cells may be formulated into sterile injectable aqueous or oleaginous suspensions according to methods known in the art and using suitable dispersing or wetting agents and/or suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parent acceptable diluent or solvent.

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

In the present invention, the term "subject" may be a mammal, 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 need of treatment. In some embodiments, the "subject" may be a clinical patient, a clinical trial volunteer, a laboratory animal, or the like. The subject may be suspected of having a disease characterized by cellular proliferation or having a disease characterized by cellular proliferation, diagnosed as having a disease characterized by cellular proliferation, or a control subject that is confirmed to not have a disease characterized by cellular proliferation, as described herein, diagnostic methods for a disease characterized by cellular proliferation and clinical demarcations of such diagnosis are known to those of skill in the art.

The pharmaceutical composition of the invention can be used for treating tumors. In the present invention, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumor (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, and melanoma. "tumor cells" may further include "solid tumors", which refer to tumors selected from the group consisting of: gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatic carcinoma), anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

The main advantages of the invention include:

(1) After the NKG2D on the surface of the engineering TIL cell recognizes the NKG2DL on the surface of the tumor cell, the NKG2D can be effectively activated by the fusion protein of the DAP10 and the CD3 zeta, so that the TIL cell can effectively kill the tumor cell.

(2) Endogenous NKG2DL expressed when the engineering TIL cells are activated can be detained in the cells by the detaining fusion protein, so that the mutual killing among the TIL cells is avoided, the brittleness in the TIL cell culture process is prevented, and the activity rate of the TIL cells is improved.

(3) The engineered TIL cell not only can identify tumor cell MHC-I molecule presenting antigen peptide to kill tumor cells through TCR, but also can kill tumors through NKG2D-NKG2DL approach, thus increasing the broad-spectrum identification capability of traditional TIL cells to tumor cells, enhancing the killing capability of TIL cells and effectively preventing the immune escape and recurrence of solid tumors.

(4) The engineering TIL cell of the invention can also express the fusion protein of the membrane-bound IL-15 which constructs the CD86 intracellular domain, so that the dryness and the killing durability of the TIL cell are obviously improved.

(5) The engineered TIL cells of the invention can induce expression of the fusion proteins of the invention by hypoxia responsive promoters in a hypoxic environment that is indicative of a solid tumor.

(6) The engineering TIL cell of the invention can also express an immune molecule switch, such as hEGFRt, so that the potential toxicity of the continuous expression of the exogenous protein is avoided, and the safety of clinical treatment is improved.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and fractions are weight percentages and weight fractions unless otherwise indicated.

The sequence information involved in the following examples is shown in table a:

nucleotide or amino acid sequences of elements of Table A

Example 1: preparation of TIL cells containing the Gene of interest

1. Plasmid construction

CDS sequences for IL-15, DAP10, NKG2D, CD3ζ and the third and fourth domains of EGFR extracellular domain were obtained at NCBI and partial sequences were codon optimized.

Four constructs 055, 073, 076, 083 were constructed separately as shown in FIG. 2. The molecular sequence of each construct was synthesized by gold Style, and the synthesized gene sequence was cloned into the basic vector pLV-EF1a-c-MYC-IRES-EGFP (purchased from Wuhan vast) to give plasmids 055, 073, 076 and 083.

2. Transformation and plasmid extraction

Trans109 competent cells were removed from-80℃and rapidly inserted into ice, after thawing, the plasmids of interest (055, 073, 076, 083) were added and mixed well and allowed to stand in ice for 25 minutes. The mixture was heat-shocked in a 42℃water bath for 45 seconds, quickly put back on ice and left to stand for 2 minutes. Mu.l of sterile LB medium without antibiotics was added, and after mixing, resuscitated at 37℃for 70 minutes at 200 rpm. 5000 The cells were collected by centrifugation at rpm for 1 minute and spread on LB medium containing the corresponding antibiotics, and placed in an incubator at 37℃overnight. The monoclonal was picked up and added to 1ml of LB medium containing antibiotics at 37℃and 200 rpm for 2 hours. The culture broth was grown overnight. 150 The cultured bacterial liquid was centrifuged at 5,000 g for 10 minutes under an overnight condition, and the supernatant was dried as much as possible to collect the bacterial cells. The bacterial pellet was resuspended with 14ml solution P1 to complete suspension. The cells were sufficiently lysed by adding 14 and ml of the solution P2. Add 14ml pre-chilled solution P3 until a yellow flocculent precipitate appears and centrifuge for 5 minutes. The filtrate was collected, and 14ml of the plasmid DNA conjugate was added to the filtrate and mixed well. Centrifuging for 2 minutes, and removing the mixed solution. 10ml of plasmid washing was added, centrifuged for 2 minutes, and the waste solution was discarded. 10ml of plasmid DNA washing 2 (absolute ethanol added) was added, centrifuged for 2 minutes, and the waste solution was discarded. The above steps are repeated. The residual ethanol was removed and the plasmid DNA eluted. The endotoxin-free plasmid DNA was eluted by centrifugation for 1 min through an endotoxin removal column. The resulting plasmid was transferred to a 1.5ml centrifuge tube.

3. Lentivirus preparation

The plasmids pLV-HRE-mIL-15 (055, 073, 076, 083), the psPAX2 vector and the pMD2.G vector containing the mIL-15 gene obtained in the step 2 are respectively and largely purified; after mixing plasmids in proportion using Lipo3000, HEK-293T cells (100 mm dish culture) with a confluency of about 80% were transfected, the transfection system was configured as follows: (1) centrifuge tube A: opti-MEM 500. Mu.l+master plasmid 10. Mu.g+pMD2.G5. Mu.g+psPAX 2 5. Mu.g+p 3000 40. Mu.l; (2) centrifuge tube B: OPti-MEM 500. Mu.l+lipo 3000 40. Mu.l. Slowly dripping the mixed solution in the centrifuge tube B into the centrifuge tube A, and standing for 15-20 min at room temperature after mixing;

addition of transfection System to HEK-293T cellsAfter transfection of 4 h, the supernatant was discarded and 10 ml DMEM complete medium was added to each dish; placing in 37 ℃ and 5 percent CO ₂ Continuously culturing in an incubator; 48 After h, the transfected 293T cell culture supernatants were collected; centrifuging at 400g for 5 min; filtering with 0.45 mu m filter to obtain filtrate as original solution of recombinant lentivirus; concentrating lentivirus by using Utra-15 centrifugal filter device, centrifuging at 5000 rpm for 50 min; packaging the concentrated lentivirus, and storing in a refrigerator at-80deg.C for use.

4. Lentivirus titer assay

Cell counts were performed on washed, digested HEK-293T cells. The lentivirus concentrate stock was diluted 10-fold by gradient dilution (50×,500×,5000×,50000×). Taking 2.5X10 ⁵ Adding HEK-293T cells into corresponding centrifuge tubes, wherein each centrifuge tube is 500 μl of culture medium-virus-cell mixed solution, uniformly mixing, and transferring into a 24-well plate; placing in 37 ℃ and 5 percent CO ₂ After culturing 24 h in the incubator, the medium is replaced; 48 After h, HEK-293T cells after infection are collected for hypoxia treatment, and the proportion of cells positive in transgene expression is detected by using a flow cytometer, so that the virus titer is calculated. Lentivirus titer calculation mode: viral titer= (m×2.5×10) ⁵ X fold dilution)/transfected volume, where m is the proportion of cells positive for transgene expression.

5. Preparation of TIL cells

Tumor mass was placed in a 10 cm petri dish, washed with PBS; removing necrotic areas and connective tissue from tumor mass with sterile ophthalmic scissors or surgical knife, and cutting into 1-3 mm pieces ³ A small block; tumor masses (e.g., lung cancer) were cultured in RPMI medium containing 10% AB serum, 1% glutamine, 1% diabody, 6000IU/mL IL-2. Observing cells under a microscope, and changing liquid every other day if no obvious adherent cells exist; if the lymphocytes are not gradually increased or the cancer cells are not reduced, continuing to change the liquid; if lymphocyte density is obviously increased and cell clusters appear, the mixture is expanded and then placed at 37 ℃ and 5% CO ₂ The culture incubator continues to culture; culturing was continued for no more than 10 days, and TIL cell collection was performed. The collected cell suspension is filtered by a filter screen with the size of 40 mu m, and tumor blocks are removed; 1000 Centrifuging at rpm for 5 min, and adding appropriate amount of REP complete culture medium for resuspension.

6. Obtaining TIL cells after lentiviral infection

Based on lentiviral titers, TIL cells were infected at moi=10; lentiviruses (moi=10), lentiboost (100×), TIL cells (3.5×10) ⁵ ) Adding into REP culture medium, mixing with 200 μl culture medium-virus-cell mixture, transferring into 48-well plate, placing at 37deg.C and 5% CO ₂ Culturing in an incubator; after 24 h infection, cells were collected, centrifuged at 1000 rpm, and virus liquid was discarded; adding appropriate amount of REP (AIM V culture medium: A1640 culture medium=1:1), resuspending the complete culture medium, transferring to 24-well plate, placing at 37deg.C, 5% CO ₂ After culturing in an incubator and culturing 72 h, the cells were collected.

Taking 1×10 ⁶ The cells were transferred to a new 24-well plate and then placed in an anoxic chamber (oxygen concentration 1%) to perform anoxic treatment (experimental group); the remaining TIL cells (as a control group without hypoxia) were transferred to a new 24-well plate and placed at 37℃in 5% CO ₂ Culturing in an incubator. The TIL cells obtained by infection with lentivirus comprising the 055, 073, 076, 083 constructs were designated 055TIL, 073TIL, 076TIL and 083TIL, respectively.

Example 2: flow detection of TIL Positive Rate of lentiviral infection

The cells prepared in example 1 were each divided into two parts: the whole negative group and the group to be tested each had a cell volume of 50. Mu.l, i.e.2.5X10 ⁵ A cell; IL-15 antibody (A09D 21-9E, beijing Baixin Biotech Co., ltd.) was added to the test group and incubated at room temperature for 15min; after the incubation, 1 ml PBS containing 2% FBS was added and 400g was centrifuged for 5min; the supernatant was discarded, 50 μl of a FITC goat anti-human IgG fcγ antibody (BioLegend, 398006) antibody cocktail formulated with PBS containing 2% FBS was added to the cells of the group to be tested, and incubated at room temperature for 10 min; 1 ml PBS containing 2% FBS was added and 400g was centrifuged for 5min; discarding the supernatant, adding 50 mu l of 7-AAD mixed solution prepared by PBS containing 2% FBS into the cells of the group to be detected, and incubating for 7 min at room temperature; adding 1 ml PBS containing 2% FBS into the whole negative group and the group to be detected, and centrifuging 400g for 5min; the supernatant was discarded, 200 μl of PBS containing 2% FBS was added to resuspend the cells, and the cells were checked on-machine.

The detection results are shown in FIG. 3. The positive rates of flow data show that 055, 073, 076, 083TIL are respectively: 30.8%, 24.5%, 27.7%, 32.7%, all exhibited higher positive rates, with the positive rate of 083TIL (32.7%) being unexpectedly higher than the other TILs.

Example 3: detection of proliferation Capacity and Activity of TIL cells

Each group of TIL cells and trophoblast cells (IL-21 NK cell expansion reagent, winning organism, cat# ZY-NKZ-0104) were cultured at 1:25 to adjust the cell density to 5X 10 ⁵ Cells/ml, cultured with REP medium (RPMI 1640: aim-v=1:1), 6-well plate, 2 ml/well, cell count every two days; fresh medium was supplemented to adjust cell density to 5×10 ⁵ Cell count, viability and dryness were determined by continuous culture at cells/ml for 14 days.

The proliferation potency test results are shown in FIG. 4. The proliferation factors of control TIL, 055TIL, 073TIL, 076TIL and 083TIL were 220, 203, 57, 167 and 198 times respectively in culture for 14 days. The proliferation capacities of 055TIL and 083TIL are basically consistent with those of the control TIL, the proliferation times of 076TIL are slightly reduced, and the proliferation times of 073TIL are obviously reduced and are far lower than those of other groups of TIL.

The results of the cell viability assay are shown in FIG. 5. The activity rates of the control TIL, the 055TIL and the 083TIL are high and are respectively 95%, 94% and 94%;076TIL activity was slightly reduced by 85%;073TIL activity was significantly reduced and cell expansion was severely affected with only 71% viability.

Example 4: detection of Stem cell fraction of TIL cells

Each group of cells prepared in example 1 was taken and divided into two parts: the whole negative group and the group to be tested each had a cell volume of 50. Mu.l, i.e.2.5X10 ⁵ A cell; 50 μl of Alexa Fluor 700 anti-human CD45RA antibody (BioLegend, 304120), APC anti-human CD62L antibody (BioLegend, 304810) and antibody mixture prepared by PBS containing 2% FBS were added to the group to be detected, and incubated at room temperature for 8 min; adding 50 μl of 7-AAD mixed solution prepared by PBS containing 2% FBS into the cells of the group to be detected, and incubating for 7 min at room temperature; adding 1 ml PBS containing 2% FBS into the whole negative group and the group to be detected, and centrifuging 400g for 5 min; the supernatant was discarded and 200 μl PBS containing 2% FBS was added to resuspend cellsAnd (5) detecting on the machine.

The detection results are shown in FIG. 6. The proportion of stem T cells was indicated by the proportion of CD45RA and CD62L double positive cells, which increased from 6.37% to 11% (055 TIL), 10.3% (076 TIL), 9.71% (083 TIL), respectively, relative to uninfected TIL cells (control TIL).

This demonstrates that the membrane-bound IL-15 constructs of the invention significantly increase the proportion of stem T cells in TIL cells. In addition, the inclusion of the membrane-bound DAP10-CD3 zeta fusion protein had substantially no effect on the proportion of dry T cells.

Example 5: detection of IFN-gamma secretion from TIL cells

Tumor cells=3:1 were co-cultured with CALU6, HELA, HCC827, which highly expressed NKG2DL, respectively, for 18 hours, and supernatants were collected and assayed using IFN-. Gamma.ELISA assay kit (human IFN-. Gamma.prefabricated ELISA kit, dake, 2307-3) according to kit instructions: sample adding: adding diluted cytokine standard to a standard hole, diluting a sample with a dilution buffer R (1×), and adding the sample to the sample hole, wherein the sample is 100 μl/well. Cover the sealing plate membrane and incubate for 2 hours at room temperature. Washing the plate: buckling and removing liquid in the holes, and adding 1X washing buffer working solution, wherein the volume is 300 [ mu ] l/well; after 1 minute of residence, the liquid in the wells was discarded. The procedure was repeated 3 times, each time with a filter paper for drying. Adding a detection antibody: biotinylated antibody working fluid, 100 μl/well, was added. Cover the sealing plate membrane and incubate for 1 hour at room temperature. The plate was repeatedly washed. Adding enzyme: and adding streptavidin-HRP working solution, wherein the ratio is 100 mu l/well. The plate was covered and incubated at room temperature (18-25 ℃) for 30 minutes. The plate was repeatedly washed. Color development: TMB,100 μl/well, was added, incubated at room temperature for 5-30 min in the dark, and termination was determined by the depth of color (dark blue) in the wells. Usually, the color development can be carried out for 10-20 minutes to achieve good effect. Terminating the reaction: and rapidly adding a stop solution, namely 100 mu l/well, and stopping the reaction. The OD value is read by a microplate reader at 450 nm. The data were analyzed by four-parameter method.

The results are shown in FIG. 7 and tables 1-3. The secretion amount of IFN-gamma after 076TIL and 083TIL are co-cultured with various tumor cells is far higher than that of uninfected control TIL and 055TIL.

TABLE 1 IFN-gamma secretion (pg/ml) for each group of TIL molecules co-cultured with CALU6 cells

TABLE 2 IFN-gamma secretion amounts (pg/ml) for groups of TIL molecules co-cultured with HELA cells

TABLE 3 IFN-gamma secretion amounts (pg/ml) for the respective groups of TIL molecules co-cultured with HCC827 cells

The above results demonstrate that 076TIL and 083TIL expressing the membrane-bound DAP10-CD3 zeta fusion proteins of the present invention not only recognize tumor cells by TCR but also activate TIL cells more efficiently by the NKG2D-NKG2L pathway than 055TIL recognizing tumor cells by TCR alone. Meanwhile, 083 prevents the brittleness of TIL through endoplasmic reticulum retention technology, so that the activity rate of 083TIL is higher than 076TIL, and is more easily activated in the process of co-culturing with target cells, thereby representing higher IFN-gamma secretion.

Example 6: results of in vitro killing experiments for each TIL

Taking 1×10 ⁵ Each of the tumor cells Calu-6 (ATCC, HTB-56 ™), HELA (ATCC, CRM-CCL-2 ™), HCC827 (CRL-2868) and A-375B 2M KO (Guangzhou Source well, cat# YKO-H486) was inoculated into 24 well plates. After 24h, TIL cells were diluted to a density of 1.5X10 ⁶ /ml. The culture medium in the original wells of the 24-well plate was pipetted off. 1ml of TIL cell fluid was added to each well. After 24 hours, the culture medium in the well was aspirated, and the cell supernatant was centrifuged for ELISA detection. The cells in the wells were washed with PBS and the tumor cell activity was detected by CCK8 method.

After 24 hours of co-culture of each group of TILs with tumor cells (HELA) in a 3:1 ratio, microscopic observation and photographing were performed. The results are shown in FIG. 8: the HELA, HELA and control TIL co-culture experimental group has good growth state of HELA cells, the cells grow in a compact single layer, HELA+055TIL experimental group HELA cells grow slowly, the cells cannot grow into cell culture holes, and part of cells become round and fall off; HELA+076TIL and 083TIL experimental groups HELA cells were rounded and shed over a large area.

The results of testing the killing activity of HELA tumor cells at E: t=3:1 for each set of TILs are shown in fig. 9 and table 4, with 076TIL and 083TIL significantly increased in killing capacity of tumor cells by about 3-fold as compared to control TILs and 055 TIL.

TABLE 4 viable cell fraction (%)

The killing effect of each group of TILs on A-375B 2M KO tumor cells at E:T of 1:1, 3:1 and 5:1, respectively, is shown in FIG. 10 and Table 5. The modified 076, 083TIL has remarkable killing effect on melanoma cell line A375 (B2M KO) relative to 055TIL and unmodified control TIL, and in a dose-dependent manner, the killing is more obvious under the condition of high-efficiency target ratio, and the cytotoxicity is about 60% (100 percent minus the proportion of living cells).

TABLE 5 Living cell proportions (%)

Example 7: antibody dependent cellular cytotoxicity Assay (ADCC)

ADCC (antibody dependent cell-mediated cytotoxicity) refers to the binding of the Fab fragment of an antibody to an epitope on the surface of a target cell, and its Fc fragment binds to FcR on the surface of a killer cell (NK cell, macrophage, neutrophil, etc.), mediating the direct killing of the target cell by the killer cell. EGFR binds to cetuximab, which causes target cell death by ADCC, rituximab does not bind to EGFR, and does not cause cell death by ADCC, thus allowing control to demonstrate the specificity of the design.

The NK cell culture kit (available from Fujian Sanyi blood manufacturing technologies Co., ltd., CT-001) was used as describedBook method NK cells were isolated from peripheral blood of healthy volunteers, amplified and labeled with CytoTell Blue. Target cells (TIL infected with 076 LV) were resuspended in medium to adjust the cell density to 5×10 ⁵ Cells/ml, 1ml each was added to 3 wells of a 6-well plate. Cetuximab (merck, germany) was added separately, rituximab (rituximab, rogowski diagnostics, gmbH, H0334) to a concentration of 200 μg/ml, PBS in the same volume, and incubated at room temperature for 40 minutes after mixing. Resuspension of NK cells with medium, adjustment of cell density to 5X 10 ⁶ 1mL of each of the above 3 wells is added into a 6-well plate, so that the effective target ratio reaches 10:1, and the final concentration of cetuximab and rituximab in the system is 100 mug/mL, and the total volume of the system is 2mL. After the system is evenly mixed, the mixture is placed in an incubator for normal culture for 24 hours. Changes in apoptosis levels (PE-coupled Annexin-V apoptosis assay kit, bi di biopharmaceutical, 559763) were detected after 24 hours.

The results are shown in FIG. 11. The apoptosis rate of 076TIL added into NK cells and rituximab (recognizing CD 20) is 5.5-8.1%, and is at a relatively low level, and the apoptosis rate of 076TIL is about 30% under the condition that NK cells and cetuximab (recognizing EGFR) coexist. Experimental results show that hRGFRt can play a specific molecular switch role, so that the safety of clinical use is improved.

Discussion of the invention

The present inventors have provided for the first time a novel and unique membrane-bound fusion protein of DAP10 and CD3 zeta. The membrane-bound DAP10-CD3 zeta fusion proteins of the invention unexpectedly effectively initiate the activation of TIL cells, thereby more effectively killing tumor cells.

In the invention, after the engineered TIL cells recognize NKG2DL on the surface of tumor cells through NKG2D on the surface, the TIL cells can be further effectively activated through the fusion protein of the invention, so that the TIL cells can effectively kill the tumor cells.

Taking 083TIL as an example, it utilizes a hypoxia inducible promoter to regulate expression of the DAP10 and CD3 zeta fusion proteins of the present invention. When TIL cells reach or around tumor tissues, the membrane-bound DAP10-CD3 zeta fusion protein is expressed, and T activation can be started through DAP10 and CD3 zeta functional elements after tumor cells are identified through NKG2D-NKG2DL, so that the tumor cells are effectively killed.

The modified TIL cell not only can identify tumor cell MHC-I molecule presenting antigen peptide to kill tumor cells through TCR, but also can kill tumors through NKG2D-NKG2DL path, and can effectively prevent immune escape and recurrence of solid tumors.

In addition, the invention also provides a detention type fusion protein with novel and unique structure, and experiments show that the detention type fusion protein can be effectively detention by an endoplasmic reticulum, so that NKG2DL endogenously expressed by TIL cells is detention in the cells, mutual killing among the TIL cells is avoided, brittleness in the TIL cell culture process is prevented, and the activity rate of the TIL cells is remarkably improved.

In addition, the engineering TIL cell of the invention can further express the IL-15 with novel and unique membrane binding structure, thereby further improving the dryness of the TIL cell.

In addition, the engineering TIL cell can further express a hEGFRt molecular switch (such as 076 TIL), can specifically cause related TIL apoptosis through ADCC action, ensures the clinical use safety of TIL treatment, and can also utilize cetuximab for flow detection as a positive rate index.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. An engineered TIL cell that enhances the ability to recognize and kill a tumor, wherein the engineered TIL cell expresses an exogenous fusion protein selected from the group consisting of: an activated fusion protein, a retentive fusion protein, or a combination thereof;

wherein the activated fusion protein is a membrane-bound DAP10-CD3 zeta fusion protein;

the retention-type fusion protein comprises: NKG2D extracellular domain and KDEL sequences.

2. The engineered TIL cell of claim 1, wherein the activated fusion protein comprises: (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 cd3ζ;

wherein the extracellular domain and intracellular signaling domain are connected in series by the transmembrane domain.

3. The engineered TIL cell of claim 1, wherein said engineered TIL cell has said activated fusion protein on a cell membrane; and/or said engineered TIL cells have said retention-type fusion protein in the endoplasmic reticulum or golgi apparatus.

4. The engineered TIL cell of claim 1, wherein said engineered TIL cell comprises a polynucleotide encoding said activated fusion protein and/or said resident fusion protein.

5. The engineered TIL cell of claim 1, wherein the engineered TIL cell comprises a nucleic acid construct comprising a first expression cassette comprising a polynucleotide encoding the activated fusion protein and/or a polynucleotide encoding the resident fusion protein.

6. The engineered TIL cell of claim 1, wherein the engineered TIL cell comprises a vector comprising: polynucleotides encoding the activated fusion protein and/or polynucleotides encoding the retentate fusion protein.

7. The engineered TIL cell of claim 1, wherein the engineered TIL cell further expresses a membrane-bound IL-15 fusion protein comprising the following elements:

(i) Interleukin 15 or a functionally active fragment thereof;

(ii) A transmembrane domain; and

(iii) An intracellular domain;

wherein the intracellular domain is a CD86 intracellular domain.

8. A composition comprising the engineered TIL cells of claim 1 and a pharmaceutically acceptable carrier.

9. A kit comprising the engineered TIL cells of claim 1, or a reagent for preparing the engineered TIL cells of claim 1, wherein the reagent is selected from the group consisting of:

(Y1) a polynucleotide encoding said activated fusion protein and said retentate fusion protein; or (b)

(Y2) a carrier comprising: polynucleotides encoding the activated fusion protein and/or polynucleotides encoding the retentate fusion protein.

10. Use of the engineered TIL cells of claim 1, or the kit of claim 9, in the manufacture of a medicament for the treatment of a tumor.