CN117625547B

CN117625547B - New generation chimeric antigen receptor for combined expression of GPX4 and application thereof

Info

Publication number: CN117625547B
Application number: CN202311074565.XA
Authority: CN
Inventors: 严俊杰; 黄智宏; 张曦; 韩德平
Original assignee: Guangzhou Baiji Biopharmaceutical Co ltd
Current assignee: Guangzhou Baiji Biopharmaceutical Co ltd
Priority date: 2022-08-25
Filing date: 2023-08-24
Publication date: 2024-06-28
Anticipated expiration: 2043-08-24

Abstract

The invention provides a new generation chimeric antigen receptor for combined expression of GPX4 and application thereof. In particular, the invention provides an engineered immune cell which is an autologous or allogeneic T cell or NK cell, and which has the following characteristics: (a) The engineered immune cell expresses a chimeric antigen receptor CAR, wherein the CAR targets a tumor surface marker; and (b) the engineered immune cell expresses an exogenous GPX4 protein. The GPX4 protein jointly expressed enhances the anti-death capacity, especially the anti-iron death capacity of the engineering immune cells, thereby realizing better and more continuous anti-tumor effect.

Description

New generation chimeric antigen receptor for combined expression of GPX4 and application thereof

Technical Field

The invention belongs to the field of immune cell therapy, and particularly relates to a chimeric antigen receptor for combined expression of GPX4 and an engineering immune cell.

Background

T cells are an important class of lymphocytes involved in cellular immunity, and can specifically recognize and kill tumor cells through signaling by antigen presenting cells. However, tumor cells also prevent specific recognition of T cells by reducing or losing epitopes, immunosuppression, tumor heterogeneity (i.e., differences in genotype to phenotype between tumor cells of the same malignancy among individuals of different patients or in different locations within the same patient), and the like, thereby evading the immune response of the body. Chimeric antigen receptor T cell (CHIMERIC ANTIGEN receptor T cell, CAR-T) therapy has been developed to address this problem. Specifically, the CAR molecule is an artificially designed and constructed receptor molecule, and is composed of a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory domain, an intracellular signaling domain and the like. Therefore, the CAR molecule has the functions of specifically recognizing tumor surface antigen, activating T cell killing activity, stimulating T cell proliferation and the like. The T cells of the patient are made to express the CAR molecule by harvesting T cells of the patient in culture and artificially transducing the gene of the CAR molecule. After being returned to the body of a patient, the T cells can specifically and efficiently identify and kill the tumor cells through the CAR molecules, so that the effect of treating the cancer is achieved.

In the last decade, CAR-T cell therapy has made significant progress in the efficacy of hematological neoplasms. However, CAR-T cell therapy does not work well in solid tumors. Lack of long-term persistence following adoptive transfer is one of the primary reasons for poor efficacy of CAR-T cells in solid tumors. The tumor microenvironment is very complex, severely affecting the various vital activities of T cells, and adverse tumor microenvironments even lead to T cell failure and death. The T cells gradually lose anti-tumor function in the death process, and can not effectively inhibit and control the proliferation or metastasis of cancers. Cell death is a key cause of lack of long-term persistence following adoptive transfer of CAR-T cells.

Therefore, there is an urgent need in the art to develop a CAR-T cell with a stronger anti-death capability to extend persistence of the CAR-T cell after adoptive transfer, to achieve better and more sustained cellular immune effects, especially against solid tumors.

Disclosure of Invention

The invention aims to provide a new generation chimeric antigen receptor for jointly expressing GPX4 and application thereof.

The invention also aims to provide immune cells constructed based on a new generation CAR, and a preparation method and application thereof.

In a first aspect of the invention, there is provided an engineered immune cell, said engineered immune cell being an autologous or allogeneic T cell or NK cell, and said engineered immune cell having the following characteristics:

(a) The engineered immune cell expresses a chimeric antigen receptor (CHIMERIC ANTIGEN receptor, CAR), wherein the CAR targets a tumor surface marker; and

(B) The engineered immune cells express exogenous GPX4 protein.

In another preferred embodiment, the T cells comprise αβt, γδ T cells, NKT cells, MAIT cells, or a combination thereof.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) Chimeric antigen receptor T cells (CAR-T cells);

(ii) Chimeric antigen receptor NK cells (CAR-NK cells).

In another preferred embodiment, the GPX4 protein may be constitutively expressed or inducible expressed.

In another preferred embodiment, a chimeric antigen receptor T cell (CAR-T cell) is provided, the CAR-T cell having a characteristic selected from the group consisting of:

(a) The CAR-T cells express a chimeric antigen receptor CAR, the CAR targeting a tumor surface marker; and/or

(B) When the CAR-T cells are contacted with a tumor cell or an inducer, the CAR-T cells induce expression of GPX4 protein.

In another preferred embodiment, in the engineered immune cell, the CAR and GPX4 protein are expressed in tandem.

In another preferred embodiment, in the engineered immune cell, the CAR and GPX4 protein are each expressed independently.

In another preferred embodiment, the tumor surface marker refers to a specific antigen on the tumor surface.

In another preferred embodiment, the chimeric antigen receptor is located at the cell membrane of the engineered immune cell.

In another preferred embodiment, the chimeric antigen receptor is localized to the cell membrane of the CAR-T cell.

In another preferred embodiment, the GPX4 protein is located intracellular to a CAR-T cell.

In another preferred embodiment, the GPX4 protein is derived from a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys); preferably a primate.

In another preferred embodiment, the GPX4 protein is of human origin.

In another preferred embodiment, the GPX4 protein is selected from the group consisting of: hGPX4 (human), mGPX4 (mouse), macaca mulatta GPX4 (macaque), pan troglodytes GPX (chimpanzee), nomascus leucogenys GPX4 (Bai Jia gibbon), saimiri boliviens GPX4 (bee and squirrel monkey), rhinopithecus roxellana GPX4 (golden monkey), piliocolobus tephrosceles GPX (condyloma rubrum), chlorocebus sabaeus GPX (black face green monkey), or a combination thereof.

In another preferred embodiment, the GPX4 protein has a sequence selected from the group consisting of seq id no:

(i) The sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4;

(ii) An amino acid sequence obtained by performing substitution, deletion, alteration or insertion of one or more (e.g., 1 to 4) amino acid residues, or adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to the N-terminus or C-terminus thereof based on the sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4; or (b)

(Iii) Amino acid sequences having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequences shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4;

and the amino acid sequence in (ii) or (iii) has the same or similar function as the sequence shown in (i) in that it can directly remove lipid peroxidation on cell membrane.

In another preferred embodiment, the CAR has a structure according to formula Ia or Ib:

L-ABD-H-TM-C-CD3ζ (Ia)

L-ABD1-ABD2-H-TM-C-CD3ζ (Ib)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

ABD (anti-binding domain), ABD1, ABD2 is an antigen binding domain or an active fragment thereof, and ABD may be derived from a single-chain variable region fragment (scFv) of a conventional antibody comprising a light chain and a heavy chain, or may be derived from a heavy chain variable region fragment (variable domain of HEAVY CHAIN anti-body, V _HH) of a single-domain antibody comprising only a heavy chain, or may be an extracellular domain of an immune cell surface receptor protein (such as NKG2D protein);

H is the no or hinge region;

TM is a transmembrane domain;

C is a costimulatory signaling domain;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ (including wild-type, or mutant/modification thereof);

The "-" is independently a connecting peptide or peptide bond.

In another preferred embodiment, L is a signal peptide selected from the group consisting of: CD8, GM-CSF, CD4, CD28, CD137, or a mutant/modification thereof, or a combination thereof.

In another preferred embodiment, the L is a signal peptide of the CD8 protein.

In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO. 9.

In another preferred embodiment, the ABD is an antibody single-chain variable region sequence that targets a tumor antigen.

In another preferred embodiment, the ABD is a heavy chain variable region sequence from a single domain antibody that targets a tumor antigen.

In another preferred embodiment, the ABD identifiable target is as follows: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD147, CD271, GUCY2C, CD, CD133, CD44, CD166, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, erbB2, NKG2D ligands (ligands), GP350, LMP2, LMP1, epCAM, VEGFR-1, lewis-Y, ROR1, claudin18.2, claudin6, CEACAM5, B7-H3, or combinations thereof.

In another preferred embodiment, the ABD is a CD133 scFv or NKG2D extracellular domain, targeting CD133 or NKG2D ligand, respectively.

In another preferred embodiment, the amino acid sequence of the CD133 scFv is shown in SEQ ID NO. 5.

In another preferred embodiment, the amino acid sequence of the NKG2D extracellular domain is shown in SEQ ID NO. 6.

In another preferred embodiment, the H is a hinge region selected from the group consisting of: CD8, CD28, CD137, igG, or a combination thereof.

In another preferred embodiment, the H is the hinge region of the CD8 protein.

In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO. 10.

In another preferred embodiment, the TM is a transmembrane region selected from the group consisting of histones or a mutation/modification ：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233, or a combination thereof.

In another preferred embodiment, the TM is the transmembrane region of the CD8 protein.

In another preferred embodiment, the amino acid sequence of said TM is shown in SEQ ID NO. 11.

In another preferred embodiment, the C is a co-stimulatory domain selected from the group consisting of histone proteins or a mutation/modification ：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD-1、Dap10、LIGHT、NKG2C、B7-H3、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、OX40L、2B4、TLR, or a mutation/modification thereof, or a combination thereof.

In another preferred embodiment, said C is a costimulatory domain of 4-1 BB.

In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO. 12.

In another preferred embodiment, the amino acid sequence of CD3 zeta is shown in SEQ ID NO. 13.

In another preferred embodiment, the CAR further has a human IgG Fc domain having the amino acid sequence set forth in SEQ ID No. 14.

In another preferred embodiment, the amino acid sequence of the CAR is shown in SEQ ID NO. 15 and SEQ ID NO. 16.

In a second aspect of the invention, there is provided a method of preparing an engineered immune cell according to the first aspect of the invention, comprising the steps of:

(A) Providing an immune cell to be modified; and

(B) Engineering the immune cell such that the immune cell expresses a CAR molecule and an exogenous GPX4 protein, thereby obtaining an engineered immune cell according to the first aspect of the invention, wherein the CAR targets a tumor surface marker.

In another preferred embodiment, in step (B), it includes:

(B1) Introducing a first expression cassette expressing the CAR into the immune cell; and

(B2) Introducing a second expression cassette expressing a GPX4 protein into the immune cell;

wherein step (B1) may be performed before, after, simultaneously with or alternatively with step (B2).

In another preferred embodiment, there is provided a method of making a CAR-T cell of the invention comprising the steps of:

(A) Providing a T cell to be engineered;

(B) Engineering the T cell such that the T cell expresses the CAR molecule and the exogenous GPX4 protein, thereby obtaining an engineered immune cell according to the first aspect of the invention.

In another preferred embodiment, in step (B), it includes:

(B1) Introducing a first expression cassette expressing the CAR into the T cell; and

(B2) Introducing a second expression cassette expressing a GPX4 protein into the T cell;

In another preferred embodiment, when the T cell to be engineered in step (a) has expressed a CAR, then in step (B) it comprises: (B2) introducing a second expression cassette into said T cell.

In another preferred embodiment, the transcription directions of the first and second expression cassettes are in the same direction (→→), opposite directions (→≡), opposite directions (≡).

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

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

In another preferred embodiment, when the first and second expression cassettes are located on the same vector, a third expression cassette for expressing a connecting peptide is further included between the first and second expression cassettes.

In another preferred embodiment, the linker peptide is P2A, F a or T2A.

In another preferred embodiment, the second expression cassette comprises a 5' untranslated region (5 ' utr) element of GPX4 upstream and/or a 3' utr element of GPX4 downstream.

In another preferred embodiment, the 5' UTR element has the nucleotide sequence as shown in SEQ ID NO. 7.

In another preferred embodiment, the 3' UTR element has the nucleotide sequence as shown in SEQ ID NO. 8.

In another preferred embodiment, the vector is a viral vector, preferably the viral vector contains the first and second expression cassettes in tandem.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

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

In another preferred embodiment, the carrier further comprises an element selected from the group consisting of: promoters, transcription enhancing elements WPRE, long terminal repeat LTR, and the like.

In another preferred embodiment, the method further comprises the step of performing a functional and validity test on the obtained engineered immune cells.

In a third aspect of the invention, there is provided a formulation comprising: the engineered immune cell of the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation contains the CAR-T cells of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the formulation is selected from the group consisting of: injection and freeze-dried preparation.

In another preferred embodiment, the formulation comprises 0.01 to 99.99% of the engineered immune cells according to the first aspect of the invention and 0.01 to 99.99% of a pharmaceutically acceptable carrier, diluent or excipient, said percentages being mass percentages of the pharmaceutical composition.

In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1X 10 ³-1×10⁸ cells/ml, preferably 1X 10 ⁴-1×10⁷ cells/ml.

In a fourth aspect of the invention there is provided the use of an engineered immune cell according to the first aspect of the invention for the preparation of a medicament or formulation for the prophylaxis and/or treatment of cancer or tumour.

In another preferred embodiment, there is provided the use of a CAR-T cell according to the invention for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.

In another preferred embodiment, the formulation contains CAR-T cells, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the tumor includes a hematological tumor and a solid tumor.

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: pancreatic cancer, breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer, or combinations thereof.

In another preferred embodiment, the tumor is selected from the group consisting of: colorectal cancer, breast cancer, liver and gall cancer.

In a fifth aspect of the invention there is provided the use of an engineered immune cell as described in the first aspect of the invention, or a formulation as described in the third aspect of the invention, for the prevention and/or treatment of cancer or tumour.

In a sixth aspect of the invention there is provided a method of treating a disease comprising administering to a subject in need of treatment an effective amount of an engineered immune cell according to the first aspect of the invention, or a formulation according to the third aspect of the invention.

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

In another preferred embodiment, the engineered immune cells contained in the engineered immune cells or formulations are cells derived from the subject (autologous cells).

In another preferred embodiment, the engineered immune cells or the engineered immune cells comprised in the preparation are cells derived from a healthy individual (allogeneic cells).

In another preferred embodiment, the method can be used in combination with other therapeutic methods.

In another preferred embodiment, the other treatment methods include chemotherapy, radiation therapy, targeted therapy, and the like.

In a seventh aspect of the invention there is provided a kit for preparing an engineered immune cell according to the first aspect of the invention, the kit comprising a container, and within the container:

(1) A first nucleic acid sequence comprising a first expression cassette for expressing the CAR; and

(2) A second nucleic acid sequence comprising a second expression cassette for co-expressing a GPX4 protein.

In another preferred embodiment, the first and second nucleic acid sequences are independent or linked.

In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

In another preferred embodiment, the first and second nucleic acid sequences are located on the same or different vectors.

In another preferred embodiment, the first and second nucleic acid sequences are located in the same vector.

In another preferred embodiment, when the first and second nucleic acid sequences are located in the same vector, a third nucleic acid sequence comprising a third expression cassette for expression of the connecting peptide is further included between the first and second nucleic acid sequences.

In another preferred embodiment, the linker peptide is P2A, F a or T2A.

In another preferred embodiment, the connecting peptide upstream contains a cleavage site for Furin protease and/or a connecting short peptide.

In another preferred embodiment, the second nucleic acid sequence further comprises a 5' untranslated region (5 ' UTR) element of GPX4 upstream of the second expression cassette and/or a 3' UTR element of GPX4 downstream of the second expression cassette.

In another preferred embodiment, the vector is a viral vector, preferably the viral vector contains the first and second nucleic acid sequences in tandem.

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

Figure 1 shows the CAR molecular structure of CD133 CAR, NKG2D CAR.

Figure 2 shows the expression levels of GPX4 in each CAR-T cell.

Figure 3 shows the in vitro killing effect of each CAR-T cell.

Detailed Description

Through extensive and intensive research, the inventor develops a chimeric antigen receptor and an engineering immune cell which jointly express GPX4 for the first time through a large number of screening, and provides a corresponding preparation method and application. The GPX4 protein expressed in a combined way endows the engineering immune cells with stronger anti-death capability, especially iron-death capability, thereby realizing better and more continuous anti-tumor effect. The present invention has been completed on the basis of this finding.

The present invention will be described in detail with respect to the engineering immune cells of the present invention, typically by taking CAR-T cells as an example. The engineered immune cells of the invention are not limited to the CAR-T cells described in the context, but have the same or similar technical features and benefits as the CAR-T cells described in the context. Specifically, when the immune cells express the chimeric antigen receptor CAR, the NK cells are identical to T cells (or the T cells may be replaced with NK cells).

Terminology

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …" or "consisting of …".

The terms "transduction," "transfection," "transformation," and the like as used herein refer to the process of delivering an exogenous polynucleotide (nucleic acid molecule) to a host cell, transcription and translation to produce a polypeptide product, including the use of plasmid molecules to introduce the exogenous polynucleotide into the host cell.

Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. Furthermore, the invention provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

"Gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce an RNA or protein product of the gene.

The term "administering" refers to physically introducing a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion.

As used herein, the terms "solid tumor", "solid tumor" are used interchangeably to refer to a shaped tumor that is reached by clinical examination such as radiographs, CT scans, B-mode ultrasound, or palpation.

As used herein, the terms "CAR", "chimeric antigen receptor" are used interchangeably and refer to the chimeric antigen receptor according to the first aspect of the invention.

It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.

Antigen binding domains

As used herein, "antigen binding domain," "single chain antibody fragment" refers to Fab fragments, fab 'fragments, F (ab') ₂ fragments, single Fv fragments, or V _HH fragments from single domain antibodies, or the extracellular domain of an immune cell surface receptor protein (e.g., NKG2D protein) having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically an scFv (single-chain variable fragment) or V _HH (variable fragment of HEAVY CHAIN anti). The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain.

Preferably, in the present invention, the scFv is an antibody single chain variable region sequence that targets a tumor antigen.

Preferably, in the present invention, the V _HH is a heavy chain variable region sequence that targets a tumor antigen.

Preferably, V _HH described herein is CD 133V _HH, targeting CD133.

Typically, the antigen binding domain of the invention is a CD133 scFv or NKG2D extracellular domain, targeting CD133 or NKG2D ligand, respectively.

NKG2D possesses a variety of ligands, NKG2D ligands include: human MHC-class I chain related molecules (i.e., MICs, such as MICA and MICB) and human UL16 binding proteins (ULBPs, also known as human RAET 1), and the like.

In normal humans, MIC distribution is limited to only gastrointestinal epithelium, endothelial cells, and fibroblasts. MIC expression is found in infection, tumor and organ transplant recipient tissue cells. Studies have demonstrated the production of MIC, which is considered a tumor-associated antigen, in many tumors of epithelial origin, such as breast, lung, ovarian, colon, glioma and melanoma.

UL16 binding protein (ULBPs, also known as human RAET 1) is a transmembrane glycoprotein expressed by human cytomegalovirus infected cells and reported members are ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6. Compared with MIC, ULBP expression is more extensive, and is expressed in a variety of normal tissues and tumors. ULBP plays an important role in the process of monitoring the escape of human cytomegalovirus from immune systems during viral infection.

CAR-T therapy

The concept of CAR-T therapy was first proposed in 1989, undergoing thirty years of development and multiple rounds of technological alternation. The first generation of CAR-T has only a single-chain antibody as an extracellular antigen binding domain and CD3 ζ as an intracellular signaling domain, and cannot fully activate the activity of T cells, resulting in poor therapeutic effects. The second generation CAR-T introduces a co-stimulatory domain based on the first generation CAR-T, thereby improving the in vitro proliferation capacity and cytokine release level of T cells. The third generation CAR-T adds a co-stimulatory domain based on the second generation CAR-T, and although the killing activity of T cells can be improved, the excessive release of cytokines can be induced. The fourth generation CAR-T expresses other auxiliary factors on the basis of the second generation CAR-T in a combined way, for example, the SAAT3/5 binding domain in the IL-12 or IL-2 Rbeta cell is expressed in a combined way, and the effects of improving the tumor killing activity, safety and the like are facilitated.

CAR-T cell therapy has made significant progress in the efficacy of hematological tumors, but has not been effective in solid tumors. Even in hematological tumors, patients who respond to CAR-T therapy remain at risk of relapse due to a variety of factors including poor T cell expansion and lack of long-term persistence following adoptive transfer. This problem is more pronounced in solid tumors, as the tumor microenvironment has a negative impact on T cell survival, infiltration and activity. While this major disadvantage of lack of long-term persistence following adoptive transfer of CAR-T cells, a key cause of this phenomenon is cell death. The causes of cell death are various, with excessive Reactive Oxygen Species (ROS) levels being one of the causes of T cell death. Excessive ROS cause lipid peroxidation of cell membranes and iron death of cells, which can result in T cells not being effectively antitumor, and are highly likely to affect the antitumor function of CAR-T cells.

The current methods for increasing the persistence of CAR-T cells are: (1) Humanizing a murine CAR molecule can reduce the immunogenicity of the CAR molecule to enhance persistence; (2) In addition, the co-stimulatory molecules CD28, ICOS, CD27, 4-1BB, OX40 and CD40L play a key role in the persistence and efficacy of CAR-T cells; (3) Fourth generation CAR-T cells have recently been developed to combat the immunosuppressive environment in Tumor Microenvironment (TME) while overcoming immune depletion. TRUCKS is designed to combine the cytotoxic function of CAR-T cells with the in situ delivery of cytokines with immunomodulatory capacity. Under the influence of the induction system, after CAR binding to antigen, cytokines are synthesized and act in an autocrine manner to increase T cell survival and expansion. Cytokines can also act by paracrine means, modulating the surrounding environment, interfering with immunosuppressive cytokines present in TME. A series of cytokines including IL-12, IL-7, IL-15, IL-18, IL-21 and IL-23 are currently under investigation and have entered the early stage of clinical trials.

Despite significant advances in CAR-T cell therapy over the past decade, the limited persistence of CAR-T cells in patients remains a challenge, mainly because T cells are difficult to adapt to various stimuli of the tumor microenvironment. These methods are directed to how to inhibit cell depletion, but do not effectively solve the effect of cell death on T cell efficacy. Inhibition of CAR-T cell death is also a method of prolonging its persistence in vivo and inhibition of cellular cells is effective in avoiding adverse effects on cellular immune efficacy caused thereby, which is a long-felt matter in the industry. The GPX4 is over-expressed on the CAR-T cells to enhance the iron death resistance of the CAR-T cells, so that the persistence of the CAR-T cells after adoptive transfer is prolonged, and the reduction of immune curative effect caused by cell death can be effectively avoided. Surprisingly, the CAR-T cells constructed in accordance with the present invention that jointly express GPX4 have not only a reduced immune response but also unexpectedly have a significant boost (manifested as a stronger killing/inhibition effect on tumor cells).

GPX4 protein

GPX4 (Glutathione Peroxidase 4), glutathione peroxidase 4, also known as phospholipid hydrogen peroxide glutathione peroxidase (PHGPx), is the fourth member of the selenium-containing GPX family. GPX4 is able to directly scavenge lipid peroxides from cell membranes and plays a key role in inhibiting cellular iron death.

Iron death (Ferroptosis) is an iron-dependent, novel, apoptosis-dependent manner, distinguished from apoptosis, cell necrosis, autophagy. The main mechanism of iron death is to catalyze unsaturated fatty acid on cell membrane to generate lipid peroxidation under the action of ferrous iron or ester oxygenase, thereby inducing cell death; furthermore, the expression level of the antioxidant system (glutathione-GSH and glutathione peroxidase 4-GPX 4) was also reduced.

Typically, the GPX4 protein of the invention has a sequence selected from the group consisting of:

(i) The sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4; (ii) An amino acid sequence obtained by performing substitution, deletion, alteration or insertion of one or more (e.g., 1 to 4) amino acid residues, or adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to the N-terminus or C-terminus thereof based on the sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4; or (iii) an amino acid sequence having at least greater than or equal to 65%, at least greater than or equal to 70%, at least greater than or equal to 75%, at least greater than or equal to 80%, at least greater than or equal to 85%, at least greater than or equal to 90%, at least greater than or equal to 95%, at least greater than or equal to 96%, at least greater than or equal to 97%, at least greater than or equal to 98%, or at least greater than or equal to 99% of the sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4; and the amino acid sequence in (ii) or (iii) has the same or similar function as the sequence shown in (i) in that it can directly remove lipid peroxidation on cell membrane.

Preferably, the GPX4 protein of the invention is of human origin, i.e. hGPX4.

The engineering immune cells can stably express GPX4 protein, and the GPX4 protein does not adversely affect the curative effects (such as tumor inhibiting effects) of corresponding CAR-T cells, CAR-NK cells, CAR-NKT cells and the like.

Furthermore, according to reports in the prior art, overexpression of GPX4 in mouse T cells does not enhance the tumor-suppressing effect of T cells. However, the invention unexpectedly and firstly discovers that the combined expression of GPX4 has a remarkable improvement on the tumor inhibition effect of the CAR-T cells.

By jointly expressing the GPX4 protein, on one hand, the death resistance of the engineering immune cells is enhanced, so that the persistence of CART cells after adoptive transfer is prolonged; on the other hand, it has surprisingly also been found that the tumor suppression effect of the engineered immune cells of the invention is further improved, and thus better, more sustained cellular immunotherapy, especially against solid tumors, can be achieved.

Chimeric Antigen Receptor (CAR)

As used herein, a chimeric immune antigen receptor (CHIMERIC ANTIGEN receptor, CAR) includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. Extracellular domains include optional signal peptides and target-specific binding domains (also referred to as antigen binding domains). The intracellular domain includes a costimulatory domain and a cd3ζ chain moiety. When CAR is expressed in T cells, the extracellular segment recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines such as IL-2 and IFN- γ, etc., affecting the tumor cells, causing the tumor cells to not grow, to be caused to die or otherwise be affected, and causing the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the cd3ζ chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain and the CD3 zeta signaling domain.

Preferably, the CAR of the invention also expresses GPX4 protein in tandem, preferably hGPX4 protein in tandem.

Preferably, the CAR of the invention has a structure according to formula Ia or Ib:

L-ABD-H-TM-C-CD3ζ (Ia)

L-ABD1-ABD2-H-TM-C-CD3ζ (Ib)

Wherein the definition of each element is as described above.

Typically, the CAR of the invention has the structure shown in FIG. 1, and the CAR of the invention is preferably BW133-2 CAR or BN001 CAR, the amino acid sequence of which is shown as SEQ ID NO:15 or SEQ ID NO:16, respectively.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to a CAR-T cell as described in the first aspect of the invention. The CAR-T cells of the invention can be used for treating various tumors, including hematological tumors and solid tumors, such as colorectal cancer, breast cancer, liver and gall cancer and the like.

CAR-T cells have the following advantages over other T cell-based therapies: (1) the course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once completed; (3) The CAR can utilize not only tumor protein antigens but also glycolipid non-protein antigens, so that the target range of the tumor antigens is enlarged; (4) Patient autologous cells can be used to reduce the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to CAR-NK cells as described in the first aspect of the invention. The CAR-T cells of the invention can be used for treating various tumors, including hematological tumors and solid tumors, such as colorectal cancer, breast cancer, liver and gall cancer and the like.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion by non-antigen specific pathways. New functions may be obtained by engineered (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

CAR-NK cells also have advantages over autologous CAR-T cells, such as: (1) The perforin and the granzyme are released to directly kill tumor cells, and the perforin and granzyme have no killing effect on normal cells of the organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storms; (3) Is easy to expand and develop into a ready-made product in vitro. In addition, similar to CAR-T cell therapy.

Engineered immune cells

As used herein, the term "engineered immune cells" is autologous or allogeneic T cells or NK cells, and has the following characteristics: (a) The engineered immune cell expresses a chimeric antigen receptor (CHIMERIC ANTIGEN receptor, CAR), wherein the CAR targets a tumor surface marker; and (b) the engineered immune cell expresses an exogenous GPX4 protein.

The engineered immune cells of the invention include chimeric antigen receptor T cells (CAR-T cells) and chimeric antigen receptor NK cells (CAR-NK cells), wherein the T cells include αβt, γδ T cells, NKT cells, MAIT cells, or a combination thereof.

The expression of the GPX4 protein in the engineered immune cells of the invention may be constitutive or inducible. In engineered immune cells, the CAR and GPX4 proteins are expressed in tandem or each independently.

Expression cassette

As used herein, an "expression cassette" or "expression cassette of the invention" includes a first expression cassette and a second expression cassette. For example, an expression cassette of the invention may be as described in the seventh aspect of the invention, the first expression cassette comprising a nucleic acid sequence encoding the CAR. The second expression cassette expresses an exogenous GPX4 protein.

In the present invention, the GPX4 protein may be constitutively expressed or inducible expressed.

In the case of induction expression, the second expression cassette expresses a GPX4 protein when the CAR-T cell contacts a tumor cell or is activated by a corresponding inducer; thus, the second expression cassette does not express the GPX4 protein when the CAR-T cells of the invention are not contacted with tumor cells or corresponding inducers.

In one embodiment, the first and second expression cassettes further comprise a promoter and/or terminator, respectively. The promoter of the second expression cassette may be a constitutive or inducible promoter.

Preferably, the second expression cassette may further comprise a 5' untranslated region (5 ' utr) element of GPX4 upstream and/or a 3' utr element of GPX4 downstream.

In one embodiment, the 5' UTR element has the nucleotide sequence as set forth in SEQ ID NO. 7.

In one embodiment, the 3' UTR element has the nucleotide sequence as set forth in SEQ ID NO. 8.

Carrier body

The invention also provides vectors containing the expression cassettes of the invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow for long-term, stable integration of transgenes into the cell genome and replication with replication of the daughter cell genome. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells and have the advantage of low immunogenicity.

In general, the expression cassette or nucleic acid sequence of the invention may be linked downstream of the promoter by conventional procedures and incorporated into an expression vector. The vector may integrate into the eukaryotic cell genome and replicate accordingly. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The vectors of the invention can also be used in standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety.

The expression cassette or nucleic acid sequence may be cloned into many types of vectors. For example, the expression cassette or nucleic acid sequence may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, and the like.

Further, the vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al, cold Spring Harbor Laboratory, new York, 2001) and other manual of virology and molecular biology. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include at least one replication origin that functions in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Many viral-based systems have been developed and used for gene transduction of mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In one embodiment, a lentiviral vector is used. Many DNA virus systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these elements are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when an element is inverted or moved relative to another element. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.

One example of a suitable promoter is the Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebstein-Barr virus (EBV) immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can either initiate expression of the polynucleotide sequence linked to the inducible promoter when desired, or shut down expression when not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

The vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expression cells from the transfected or infected cell population by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neomycin and the like.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of vectors, the vector may be readily introduced into a host cell, e.g., a mammalian (e.g., human T cell), bacterial, yeast or insect cell, by any method known in the art. For example, the vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., molecular Cloning: A Laboratory Manual (Sambrook et al, cold Spring Harbor Laboratory, new York, 2001). Preferred methods for introducing the polynucleotide into a host cell are liposome transfection and cationic complex polyethylenimine transfection.

Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery tool (DELIVERY VEHICLE) is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. The lipid is a lipid substance, which may be a naturally occurring or synthetic lipid. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

It will be appreciated that in the present invention, instead of transduction with a plurality of lentiviruses, mRNA or plasmid may be transfected directly or by expression of artificial transcription factors or the like, to co-express GPX4 and CAR molecules in immune cells such as T cells.

Formulations

The invention provides an engineered immune cell (e.g., CAR-T cell) comprising the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-T cells in the formulation is 1 x 10 ³-1×10⁸ cells/ml, more preferably 1 x 10 ⁴-1×10⁷ cells/ml.

In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells) transduced with vectors (e.g., lentiviral vectors) comprising the expression cassettes of the invention. The transduced T cells can target the surface markers of tumor cells and express GPX4 protein, and the killing efficiency of the transduced T cells on the tumor cells is obviously improved in a synergistic way.

Accordingly, the present invention also provides a method of stimulating a CAR-T cell mediated immune response targeted to a mammalian tumor cell population or tissue, comprising the steps of: administering the CAR-T cells of the invention to a mammal.

In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This approach results in a very low probability of graft versus host response, and antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained control of tumors.

In one embodiment, the CAR-T cells of the invention can undergo stable in vivo expansion and can last from months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-T cells can induce a specific immune response to highly expressing tumor cells of the antigen recognized by the CAR antigen binding domain.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Types of cancers treated with the CARs of the invention include hematological and solid tumors, including but not limited to: colorectal cancer, breast cancer, hepatobiliary cancer, etc., wherein the hepatobiliary cancer also comprises liver cancer, gallbladder cancer, cholangiocarcinoma, etc

In general, cells activated and expanded as described herein are useful in the treatment and prevention of diseases such as tumors. Accordingly, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the invention.

The CAR-T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a formulation of the invention may comprise an engineered immune cell as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

The formulations of the present invention may be administered in a manner appropriate for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, or may be determined by clinical trials.

When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the formulation of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). Formulations comprising the CAR-T cells described herein can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably at a dose of 10 ⁵ to 10 ⁷ cells/kg body weight (including all whole values within the range). The CAR-T cell formulation may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676,1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject formulation may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the CAR-T cell formulation of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the CAR-T cell formulation of the invention is preferably administered by intravenous injection. The CAR-T cell preparation can be injected directly into a tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding CAR-T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the CAR-T cells of the invention can be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell preparation of the invention is administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, from 1 x 10 ⁵ to 1 x 10 ¹⁰ modified CAR-T cells of the invention can be administered to a patient by, for example, intravenous infusion, per treatment or per course of treatment.

The main advantages of the invention include:

1. the engineering immune cells of the invention jointly express GPX4 protein, and have stronger anti-death capability, in particular to iron-resistant death capability.

2. Compared with the second generation CAR-T, the engineering immune cell provided by the invention has stronger tumor killing effect.

3. Compared with the third generation CAR-T, the engineering immune cell has stronger safety and low probability of inducing excessive release of cytokines.

4. The engineered immune cells of the invention have greater persistence in an in vivo environment.

5. The expression of GPX4 protein in the engineering immune cells can reduce the cell death rate and enhance the anti-tumor effect of the engineering immune cells.

6. Compared with the CAR-T of the past generation, the engineering immune cell can play a better anti-tumor effect in various tumors (including hematological tumors and solid tumors), and especially aims at colorectal cancer, breast cancer, liver and gall cancer and other solid tumors.

7. According to reports in the prior literature, overexpression of GPX4 in mouse T cells does not enhance the tumor-suppressing effect of T cells. However, the invention unexpectedly and firstly discovers that the combined expression of GPX4 has a remarkable improvement on the tumor inhibition effect of the CAR-T cells.

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 parts are weight percentages and parts unless otherwise indicated.

Sequence information

Amino acid sequence of GPX4 protein transcription variant 1 (SEQ ID NO: 1)

Transcript variant 1NM_002085.5

MSLGRLCRLLKPALLCGALAAPGLAGTMCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF

Amino acid sequence of GPX4 protein transcription variant 2 (SEQ ID NO: 2)

Transcript variant 2NM_001039847.3

MSLGRLCRLLKPALLCGALAAPGLAGTMCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQUGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFGHRLSTVPHRQERLRGEALRTHGGAPGDREGPAPLFLAPQVCGPARAPAHALGAFHRHS

Amino acid sequence of GPX4 protein transcription variant 3 (SEQ ID NO: 3)

Transcript variant 3NM_001039848.4

MGRAGAGSPGRRRQRCQSRGRRRPRAPRRRKAPACRRRRARRRRKKPCPRSLRPEIHECPKSQDPCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQUGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF

Amino acid sequence of GPX4 protein transcriptional variant 4 (SEQ ID NO: 4)

Transcript variant 4NM_001367832.1

MCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQUGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF

Amino acid sequence of CD133 scFv (SEQ ID NO: 5)

DVVVTQTPLSLPVSFGDQVSISCRSSQSLANSYGNTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPYTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFSDFEMHWVKQTPVHGLEWIGDIDPGTGDTAYNLKFKGKATLTTDKSSSTAYMELRSLTSEDSAVYYCTLGAFVYWGQGTLVTVSS

The amino acid sequence of the NKG2D extracellular domain (SEQ ID NO: 6)

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

Nucleotide sequence of GPX 4' UTR (SEQ ID NO: 7)

GCCTTTGCCGCCTACTGAAGCCGGCGCTGCTCTGTGGGGCTCTGGCCGCGCCTGGCCTGGCCGGGACC

Nucleotide sequence of GPX 4' UTR (SEQ ID NO: 8)

CTCCACAAGTGTGTGGCCCCGCCCGAGCCCCTGCCCACGCCCTTGGAGCCTTCCACCGGCACTCATGACGGCCTGCCTGCAAACCTGCTGGTGGGGCAGACCCGAAAATCCAGCGTGCACCCCGCCGGAGGAAGGTCCCATGGCCTGCTGGGCTTGGCTCGGCGCCCCCACCCCTGGCTACCTTGTGGG

Amino acid sequence of human CD8 Signal peptide (SEQ ID NO: 9)

MALPVTALLLPLALLLHAARPS

Amino acid sequence of human CD8 hinge region (SEQ ID NO: 10)

TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFA

Amino acid sequence of human CD8 transmembrane domain (SEQ ID NO: 11)

CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR

Amino acid sequence of human 4-1BB intracellular domain (SEQ ID NO: 12)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Amino acid sequence of human CD3 zeta intracellular signal transduction domain (SEQ ID NO: 13)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

IgG Fc amino acid sequence (SEQ ID NO: 14)

ESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The amino acid sequence of BW133-2 CAR (SEQ ID NO: 15)

MALPVTALLLPLALLLHAARPSDVVVTQTPLSLPVSFGDQVSISCRSSQSLANSYGNTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPYTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFSDFEMHWVKQTPVHGLEWIGDIDPGTGDTAYNLKFKGKATLTTDKSSSTAYMELRSLTSEDSAVYYCTLGAFVYWGQGTLVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRBN001 CAR Amino acid sequence of (SEQ ID NO: 16)

MALPVTALLLPLALLLHAARPSIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFF

DESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSP

NLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEAS

RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMR

PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR

RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD

TYDALHMQALPPR

Materials and methods

CAR molecules and structures thereof

In an embodiment, each CAR molecule comprises the following partial structure: human CD8 signal peptide [ abbreviated CD8 (SP) ], anti-human CD133 single chain antibody [ abbreviated CD133 scFv ], human NKG2D extracellular domain [ abbreviated NKG2D (ED) ], human CD8 hinge region [ abbreviated CD8 (range) ], human IgG Fc domain [ abbreviated IgG (Fc) ], human CD8 transmembrane domain [ abbreviated CD8 (TM) ], human 4-1BB intracellular domain [ abbreviated 4-1BB (ID) ], human CD3 zeta intracellular signal transduction domain [ abbreviated CD3 zeta (ID) ].

The NKG2D CAR molecule was designated BN001 and the CD133 CAR molecule was designated BW133-2.

The structure of the CAR molecule is shown in figure 1, and is specifically as follows:

BW133-2 is composed of CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID) and CD3 zeta (ID) which are sequentially connected in series from the amino terminal to the carboxyl terminal;

BN001 is composed of CD8 (SP), NKG2D (ED), CD8 (range), CD8 (TM), 4-1BB (ID) and CD3 zeta (ID) which are sequentially connected in series from the amino terminal to the carboxyl terminal.

General method

Car-T cell iron death characterization

(1) CAR-T cell lipid peroxidation assay

Cells were cultured overnight at 37℃in a 5% CO ₂ incubator with iron death promoter (RSL 3) for experiments, while an iron death inhibitor control group (Fer-1) was designed. The supernatant was then removed and washed 2 times with PBS. 200. Mu. l Liperfluo (final concentration 5. Mu.M) in PBS was added to each well and incubated for 30min at 37℃in a 5% CO ₂ incubator. The PBS was washed 2 times. And (5) performing flow detection.

(2) CAR-T cell mortality detection

Cells were cultured overnight at 37℃in a 5% CO ₂ incubator with iron death promoter (RSL 3) for experiments, while an iron death inhibitor control group (Fer-1) was designed. The supernatant was then removed and washed 2 times with PBS. 10. Mu.l of Annexin-V (20. Mu.g/ml) marked by FITC and 100. Mu. lBinding Buffer were added, light was shielded from the room temperature for 30min, 5. Mu.l of PI (50. Mu.g/ml) was added, after 5min of light shielding reaction, 400. Mu.l of Binding Buffer was added, and flow cytometry was immediately performed while a tube without addition of Annexin V-FITC and PI was used as a negative control.

2. Target cell detection

(1) Expression detection of CD133

Cells to be detected were washed twice with PBS and resuspended with FACS buffer. FITC-labeled anti-CD 133 antibody was added to the cell suspension to be assayed according to the antibody instructions and incubated at 4℃for 60min. Target cells incubated with isotype antibody were used as negative controls and the CD133 expression rate of the target cells was measured by flow cytometry. Analysis was performed using CytExpert software.

(2) Detection of expression of NKG2D ligand

Cells to be detected were washed twice with PBS and resuspended with FACS buffer. FITC-labeled anti-NKG 2D antibody was added to the cell suspension to be detected according to the antibody instructions and incubated for 60min at 4 ℃. Target cells incubated with isotype antibody were used as negative controls and the NKG2D ligand expression rate of the target cells was measured using a flow cytometer. Analysis was performed using CytExpert software.

Example 1: lentivirus preparation

(1) Lentiviral vector plasmid transfection 293T cells

After construction of lentiviral expression vectors for BW133-2, BN001 and GPX4 (e.g., GPX4 protein transcriptional variant 4, SEQ ID NO: 4), each vector plasmid was mixed with lentiviral packaging plasmids pMD2.G, pRSV-Rev and pMDLg/pRRE, respectively, using polyethylenimine transfection reagents, and 293T cells were co-transfected. 6h after transfection was replaced with complete medium. After 72h of culture, respectively collecting virus supernatant, centrifuging at 3000rpm at 4 ℃ for 10-15 min, filtering by a filter membrane with the aperture of 0.45 mu m, concentrating the virus, and storing the collected virus concentrate at-80 ℃.

(2) Lentivirus titer assay

Mu.l of complete medium (AIM medium+5% serum replacement+100U/ml penicillin+100. Mu.g/ml streptomycin) and PBMC cells were added to each well of a 96-well plate to give a cell density of 1X 10 ⁵ cells/well. And taking the slow virus concentrated solution and carrying out gradient dilution by using a complete culture medium. Lentiviruses were added to the 96-well plates at 25 μl/well for each dilution gradient, and the lentiviruses were allowed to infect PBMC cells (human PBMC cells of the negative control group were added to complete medium only), and placed in a cell incubator for culture (culture temperature 37 ℃, carbon dioxide concentration 5%) and fluid infusion was performed on alternate days. After 3 days of culture, the cells in each well were gently mixed and transferred to a 1.5ml centrifuge tube, and virus titer was measured by qPCR.

Example 2: preparation and detection of CAR-T cells

(1) T cell preparation

Peripheral blood mononuclear cell densities of healthy donors were adjusted to 2X 10 ⁶/ml, 50ng/ml of anti-CD 3 antibody, 50ng/ml of anti-CD 28 antibody, and 200IU/ml of recombinant IL-2 were added, and cultured in a cell incubator for 24 hours (culture temperature: 37 ℃ C., carbon dioxide concentration: 5%).

(2) Lentivirus transduced T cells

The obtained T cells were washed and the cell density was adjusted to 4X 10 ⁶ cells/ml. Lentivirus was added at MOI=1 to 10IU/ml for transduction while supplementing 50ng/ml of anti-CD 3 antibody, 50ng/ml of anti-CD 28 antibody, and 200IU/ml of recombinant IL-2, and cultured in a cell incubator (culture temperature: 37 ℃ C., carbon dioxide concentration: 5%). After 24h, the cell density was adjusted to 1.5-2X 10 ⁶/ml and 300IU/ml of IL-2 was supplemented. On day 4 after transduction, cells were washed to remove residual lentiviral particles in the supernatant and cultured in a cell incubator for 5 days (culture temperature: 37 ℃ C., carbon dioxide concentration: 5%) while maintaining cell density of 1 to 2X 10 ⁶/ml. Cells were harvested on day 10 post transduction and frozen in liquid nitrogen with frozen stock (frozen medium with 5% human serum albumin: physiological saline=1:1). The CAR-T cells obtained follow the nomenclature of the corresponding CAR molecule, wherein CD133 CAR-T cells co-expressing GPX4 are named BW133-2+gpx4, NKG2D CAR-T cells co-expressing GPX4 are named BN001+gpx4, and T cells not transduced with lentivirus are named Ctrl T.

(3) Expression detection of CAR molecules

Ctrl T, BW133-2 and BW133-2+gpx4 cells to be detected were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% bsa). Human CD133 protein was incubated with CAR-T cells for 1 hour following antibody instructions, followed by centrifugation to remove the supernatant and resuspension with FACS buffer (PBS containing 0.1% sodium azide and 0.4% bsa). The anti-His tag antibody is added to the cell suspension to be detected, and the cell suspension is incubated for 60min at 4 ℃. The expression rates of CD133 CAR molecules in BW133-2 and BW133-2+GPX4 cells were measured using a flow cytometer using Ctrl T cells as a negative control, and the results are shown in Table 1.

Ctrl T, BN001 and BN001+ GPX4 cells to be detected were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% bsa). APC-labeled anti-human NKG2D antibodies were added to the cell suspension to be detected according to the antibody instructions and incubated at 4 ℃ for 30min. The expression rate of NKG2DCAR molecules in BN001 and BN001+gpx4 cells was measured by flow cytometry using Ctrl T cells as a negative control, and the results are shown in table 1.

(4) Expression detection of CAR-T cell GPX4

Ctrl T and each CAR-T cell to be detected were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% bsa). Cells were fixed according to the fixed rupture kit and anti-human GPX4 antibodies were incubated with CAR-T cells for 1.5h according to the antibody instructions. The Ctrl T and each CAR-T cell to be detected were then washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% bsa), followed by the addition of anti-rabbit secondary antibody and incubation with CAR-T cells for 1.5h. The Ctrl T and each CAR-T cell to be detected were then washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% bsa). And (3) taking Ctrl T cells as negative control, and detecting the expression rate of the CAR-T cell GPX4 molecules by using a flow cytometer.

The results are shown in table 1 and fig. 2, although there was background expression of GPX4 in T cells (the expression rate contained multiple GPX4 transcription variants), the MFI level of GPX4 in BW133-2+gpx4 cells was significantly higher than that of BW133-2 cells, and that of GPX4 in BN001+gpx4 cells was also significantly higher than that of BN001 cells, indicating that the exogenous GPX4 protein had been successfully expressed in the T cells described above (BW 133-2+gpx4 cells, BN001+gpx4 cells).

Table 1 detection of expression level of CAR molecules and GPX4 in each CAR-T cell

CAR-T cells	CAR expression Rate	GPX4 expression Rate	GPX4 MFI
				Ctrl T	0％	98.4％	25715
BW133-2	76.7％	99.8％	29651
				BW133-2+GPX4	50.9％	99.8％	33538
BN001	100％	99.7％	26680
				BN001+GPX4	100％	99.9％	30937

Example 3: CAR-T cell in vitro function experiment

HT55-mCherry cells were resuspended in corresponding complete medium at a density of 1X 10 ⁵/ml and plated in 96-well plates at a volume of 100. Mu.l per well. After the cells are placed in an IncuCyte SX5 living cell imaging analysis system for overnight culture, adding T cells according to the target ratio of 1:1 for co-culture, starting to record the killing effect of the T cells on tumor cells in real time, and respectively adding the target cells again at a proper subsequent time point so as to repeatedly stimulate the T cells. The mCherry fluorescence area change condition of the target cells in each group is calculated by using the inticutyte software, and the lower the signal value is, the lower the number of the target cells in the group is, and the better the T cell killing effect is.

For CD133 CAR-T, the results are shown in FIG. 3A, where BW133-2+GPX4 cells significantly inhibited growth of HT55 cells more than BW133-2 cells (P < 0.05). Again HT55 (fig. 3A arrow is the time point of the secondary stimulation) was added to stimulate CAR-T cells, and it was found that by the final time point, the relative fluorescence area of HT55 cells under BW133-2 cell treatment was 224.1% ± 8.4%, whereas the tumor suppression effect of BW133-2+gpx4 was further enhanced, and the relative fluorescence area of HT55 cells under treatment was 188.9% ± 9.3% (P < 0.05).

Similarly, for NKG2D CAR-T, as shown in fig. 3B, HT55 (arrow in fig. 3A is the time point of the secondary stimulation) was added to stimulate CAR-T cells, and it was found that the relative fluorescence area of HT55 cells under BN001 cell treatment was 100.7% ± 2.8%, whereas tumor suppression by b001+gpx4 was further enhanced, and the relative fluorescence area of HT55 cells under treatment was 86.0% ± 0.8% (P < 0.05).

According to reports in the prior literature, overexpression of GPX4 in mouse T cells does not enhance the tumor-suppressing effect of T cells. Thus, the present study unexpectedly found for the first time that the tumor suppression effect of co-expressing GPX4 on CAR-T cells was significantly improved.

Example 4: in situ CAR-T cell iron death characterization of tumors

(1) CAR-T cell lipid peroxidation assay

The CAR-T cells from the tumors were extracted enzymatically and incubated with 200. Mu. lLiperfluo (final concentration 5. Mu.M) in PBS for 30min in a 37℃5% CO ₂ incubator, respectively. The PBS was washed 2 times. And (5) performing flow detection.

The results show that the lipid peroxidation degree of the CAR-T cells constructed by the CAR molecules BW133-2+GPX4 and Bn001+GPX4 which are combined with the GPX4 protein is significantly lower than that of the CAR-T cells constructed by the CAR molecules BW133-2 and Bn001 which are not combined with the GPX4 protein.

The above results illustrate: expression of GPX4 protein cleared lipid peroxidation on CAR-T cell membranes, thereby inhibiting cell iron death.

(2) CAR-T cell mortality detection

CAR-T cells in tumors were extracted by enzymatic hydrolysis, washed 2 times with PBS, added with 100. Mu.l Binding Buffer and FITC-labeled Annexin-V (20. Mu.g/ml) 10. Mu.l, protected from light at room temperature for 30min, further added with PI (50. Mu.g/ml) 5. Mu.l, reacted from light for 5min, added with 400. Mu.l Binding Buffer, immediately subjected to flow cytometry detection, and one tube without addition of Annexin V-FITC and PI was used as a negative control.

The results show that the cell death rate of the CAR-T cells constructed by combining the CAR molecules BW133-2+GPX4 and Bn001+GPX4 which express the GPX4 protein is obviously lower compared with the CAR-T cells constructed by not combining the CAR molecules BW133-2 and Bn001 which express the GPX4 protein.

The above results illustrate: expression of GPX4 protein inhibited cell iron death, thereby significantly reducing CAR-T cell mortality.

According to the detection of CAR-T cell iron death characterization in example 4, it was demonstrated that expression of GPX4 protein cleared lipid peroxidation on CAR-T cell membranes and inhibited cell iron death, reducing CAR-T cell mortality, making tumor killing effect more durable.

Discussion of the invention

Very interesting is that different immune cells exhibit different sensitivities to iron death. GPX4 is a key protein for inhibiting iron death, GPX4 genes are knocked out in B1 cells and border region B cells, and the cells accumulate lipid peroxide and trigger iron death so as to influence the functions of the B cells; however, knocking out GPX4 in follicular B cells did not induce follicular B cells to undergo iron death. The possible mechanism is that the fatty acid transporter CD36 content on the cytoplasmic membrane of B1 cells and border zone B cells is significantly higher than that of follicular B cells, resulting in more fatty acid and lipid droplets in B1 and border zone B cells and thus more susceptible to lipid peroxidation.

Furthermore, similar to B cells, different macrophage subpopulations also have different sensitivities to iron death. RSL3 is widely used as an inhibitor of GPX4 to induce the onset of cellular iron death. The proinflammatory M1 macrophage has higher nitric oxide free radical in the M1 cell because of the high content of intracellular Inducible Nitric Oxide Synthase (iNOS) in the Yu Kangyan type M2 macrophage, so that lipid peroxidation is inhibited; in contrast, M2 macrophages have less nitric oxide radicals generated due to lower iNOS content and have less inhibition on lipid peroxidation. Therefore RSL3 does not induce iron death in M1 macrophages, but can induce iron death in M2 macrophages.

Gene deletion of Gpx4 or treatment with Gpx4 inhibitors (e.g., RSL3, ML162, and ML 210) can induce lipid peroxidation and iron death of T cells in vitro. In contrast, overexpression of Gpx4 and Aifm2 or knockout Acsl4 can protect cd8+ T cells from iron death. T cell specific loss of GPX4 does not affect thymic production in mice but impairs peripheral cd8+ T cell homeostasis, while cd4+ or cd8+ T cells lacking GPX4 cannot proliferate in acute infections, and this proliferation defect can be repaired by vitamin E supplementation (an effective liposoluble antioxidant).

The above results indicate that:

On the one hand, the mechanism of iron death is not only related to GPX4, but also involves many other genes/proteins/pathways/related enzymes or substrates, etc., and thus it is not obvious to expect a certain effect of inhibiting iron death by modulating (up-regulating) GPX4 alone;

On the other hand, different immune cells exhibit different sensitivities to iron death (even though the same immune cell has different fine subtypes, its sensitivity to iron death) and the regulation of GPX4 also exhibits different sensitivities, so it is unknown whether up-regulating the expression of GPX4 can produce an effect of inhibiting iron death for different immune cells without experimental verification.

Furthermore, according to reports in the prior literature, overexpression of GPX4 in mouse T cells did not enhance the tumor-suppressing effect of T cells. In the application, the combined expression of GPX4 not only improves the anti-iron death capability of immune cells, but also enhances the tumor inhibiting effect of immune cells, which is one of the unexpected effects of the technical scheme of the application.

In summary, the technical solution for the combined expression of GPX4 on immune cells (e.g. CAR-T cells) is unpredictability and innovative.

All documents mentioned in this disclosure are incorporated by reference in this disclosure 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 application as defined in the appended claims.

Claims

1. An engineered immune cell, wherein the engineered immune cell is an autologous or allogeneic T cell, and wherein the engineered immune cell has the following characteristics:

(B) The engineered immune cells express exogenous GPX4 protein.

2. The engineered immune cell of claim 1, wherein the T cell comprises an αβ T, γδ T cell, NKT cell, MAIT cell, or a combination thereof.

3. The engineered immune cell of claim 1, wherein the GPX4 protein is constitutively expressed or inducible expressed.

4. The engineered immune cell of claim 1, wherein the engineered immune cell is a CAR-T cell having a characteristic selected from the group consisting of:

5. The engineered immune cell of claim 1, wherein in the engineered immune cell, the CAR and GPX4 proteins are expressed in tandem; or in the engineered immune cell, the CAR and GPX4 protein are each expressed independently.

6. The engineered immune cell of claim 1, wherein the tumor surface markers refer to specific antigens on the tumor surface.

7. The engineered immune cell of claim 1, wherein the chimeric antigen receptor is located at a cell membrane of the engineered immune cell.

8. The engineered immune cell of claim 1, wherein the GPX4 protein is derived from a human or non-human mammal.

9. The engineered immune cell of claim 8, wherein said non-human mammal is selected from the group consisting of: rodents, primates.

10. The engineered immune cell of claim 9, wherein the rodent is selected from the group consisting of: rats and mice.

11. The engineered immune cell of claim 9, wherein the primate is selected from the group consisting of: and (3) a monkey.

12. The engineered immune cell of claim 1, wherein the GPX4 protein is selected from the group consisting of: human hGPX4, murine mGPX4, macaque Macaca mulatta GPX4, chimpanzee Pan troglodytes GPX, white cheek gibbon Nomascus leucogenys GPX4, bee and squirt monkey Saimiri boliviens GPX, sichuan monkey rhinopithecus roxellana GPX4, red wart piliocolobus tephrosceles GPX4, black face green monkey Chlorocebus sabaeus GPX, or combinations thereof.

13. The engineered immune cell of claim 1, wherein the CAR has a structure according to formula Ia or Ib:

L-ABD-H-TM-C-CD3ζ (Ia)

L-ABD1-ABD2-H-TM-C-CD3ζ (Ib)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

ABD (anti-binding domain), ABD1, ABD2 is an antigen binding domain, ABD is derived from a single chain variable region fragment (scFv) of a conventional antibody comprising a light chain and a heavy chain, or a heavy chain variable region fragment (variable domain of HEAVY CHAIN antibody, V _HH) of a single domain antibody comprising only a heavy chain, or an extracellular domain of an immune cell surface receptor protein NKG 2D;

H is the no or hinge region;

TM is a transmembrane domain;

C is a costimulatory signaling domain;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

The "-" is independently a connecting peptide or peptide bond.

14. The engineered immune cell of claim 13, wherein L is a signal peptide selected from the group consisting of: CD8, GM-CSF, CD4, CD28, CD137, or a combination thereof.

15. The engineered immune cell of claim 13, wherein L is a signal peptide of CD8 protein.

16. The engineered immune cell of claim 13, wherein the amino acid sequence of L is set forth in SEQ ID No. 9.

17. The engineered immune cell of claim 13, wherein the target recognized by ABD is selected from the group consisting of: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD147, CD271, GUCY2C, CD, CD133, CD44, CD166, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, erbB2, NKG2D ligand, GP350, LMP2, LMP1, epCAM, VEGFR-1, lewis-Y, ROR1, claudin18.2, claudin6, CEACAM5, B7-H3, or combinations thereof.

18. The engineered immune cell of claim 13, wherein said ABD is a CD133 scFv or NKG2D extracellular domain, targeting CD133 or NKG2D ligand, respectively.

19. The engineered immune cell of claim 18, wherein the amino acid sequence of the CD133 scFv is set forth in SEQ ID No. 5.

20. The engineered immune cell of claim 18, wherein the amino acid sequence of the NKG2D extracellular domain is set forth in SEQ ID No. 6.

21. The engineered immune cell of claim 13, wherein H is a hinge region selected from the group consisting of: CD8, CD28, CD137, igG, or a combination thereof.

22. The engineered immune cell of claim 13, wherein H is the hinge region of the CD8 protein.

23. The engineered immune cell of claim 13, wherein the amino acid sequence of H is set forth in SEQ ID No. 10.

24. The engineered immune cell of claim 13, wherein the TM is a transmembrane region ：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233, selected from the group consisting of the following histones, or a combination thereof.

25. The engineered immune cell of claim 13, wherein the TM is the transmembrane region of the CD8 protein.

26. The engineered immune cell of claim 13, wherein the TM has an amino acid sequence set forth in SEQ ID No. 11.

27. The engineered immune cell of claim 13, wherein C is a costimulatory domain ：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB (CD137)、PD-1、Dap10、LIGHT、NKG2C、B7-H3、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、OX40L、2B4、TLR, selected from the group consisting of histones, or a combination thereof.

28. The engineered immune cell of claim 13, wherein C is a costimulatory domain of 4-1 BB.

29. The engineered immune cell of claim 13, wherein the amino acid sequence of C is set forth in SEQ ID No. 12.

30. The engineered immune cell of claim 13, wherein the amino acid sequence of cd3ζ is set forth in SEQ ID No. 13.

31. The engineered immune cell of claim 1, wherein the CAR further has a human IgG Fc domain having an amino acid sequence as set forth in SEQ ID No. 14.

32. The engineered immune cell of claim 1, wherein the CAR has an amino acid sequence set forth in SEQ ID No. 15 and SEQ ID No. 16.

33. The engineered immune cell of any one of claims 1-32, wherein the GPX4 protein has a sequence selected from the group consisting of: the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

34. A method of preparing an engineered immune cell of any one of claims 1-33, comprising the steps of:

(A) Providing a T cell to be engineered; and

(B) Engineering the T cell such that the T cell expresses a CAR molecule and an exogenous GPX4 protein, thereby obtaining the engineered immune cell of any one of claims 1-33, wherein the CAR targets a tumor surface marker.

35. The method of claim 34, wherein in step (B), comprising:

36. The method of claim 34, wherein when the T cell to be engineered in step (a) has expressed a CAR, then in step (B) it comprises: (B2) introducing a second expression cassette into said T cell.

37. The method of claim 35, wherein the direction of transcription of the first expression cassette and the second expression cassette is co-directional (→), opposite (→≡), opposite (≡).

38. The method of claim 35, wherein the first expression cassette and the second expression cassette are on the same or different vectors.

39. The method of claim 35, further comprising a third expression cassette for expressing a linker peptide between the first and second expression cassettes when the first and second expression cassettes are located on the same vector.

40. The method of claim 39, wherein the linker peptide is P2A, F A or T2A.

41. The method of claim 35, wherein the second expression cassette comprises a 5' untranslated region (5 ' utr) element of GPX4 upstream and/or a 3' utr element of GPX4 downstream.

42. The method of claim 41, wherein the nucleotide sequence of the 5' utr element is set forth in SEQ ID No. 7.

43. The method of claim 41, wherein the nucleotide sequence of the 3' utr element is set forth in SEQ ID No. 8.

44. The method of claim 38, wherein the vector is a viral vector.

45. The method of claim 44, wherein the viral vector comprises the first and second expression cassettes in tandem.

46. The method of claim 38, wherein the carrier is selected from the group consisting of: DNA, RNA, or a combination thereof.

47. The method of claim 38, wherein the carrier is selected from the group consisting of: plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

48. The method of claim 38, wherein the vector is a pCDH-series lentiviral vector.

49. The method of claim 38, wherein the carrier further comprises an element selected from the group consisting of: promoter, transcription enhancing element WPRE, long terminal repeat LTR.

50. The method of any one of claims 34-49, further comprising the step of performing functional and validity assays on the obtained engineered immune cells.

51. A formulation comprising the engineered immune cell of any one of claims 1-33, and a pharmaceutically acceptable carrier, diluent, or excipient.

52. The formulation of claim 51, wherein the formulation is a liquid formulation.

53. The formulation of claim 51, wherein the formulation is in a dosage form selected from the group consisting of: injection and freeze-dried preparation.

54. The formulation of claim 51, which comprises 0.01 to 99.99% of the engineered immune cells of any one of claims 1 to 33 and 0.01 to 99.99% of a pharmaceutically acceptable carrier, diluent or excipient, said percentages being by mass of the pharmaceutical composition.

55. The formulation of claim 51, wherein the concentration of the engineered immune cells in the formulation is 1 x 10 ³-1×10⁸ cells/ml.

56. The formulation of claim 51, wherein the concentration of the engineered immune cells in the formulation is 1 x 10 ⁴-1×10⁷ cells/ml.

57. Use of an engineered immune cell according to any one of claims 1 to 33, or a formulation according to any one of claims 51 to 56, for the preparation of a medicament or formulation for the prophylaxis and/or treatment of cancer or tumour.

58. The use of claim 57, wherein the tumor is selected from the group consisting of: hematological tumors and solid tumors.

59. The use of claim 57, wherein the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

60. The use of claim 57, wherein the solid tumor is selected from the group consisting of: pancreatic cancer, breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer, or combinations thereof.

61. The use of claim 57, wherein the tumor is selected from the group consisting of: colorectal cancer, breast cancer, liver and gall cancer.

62. A kit for preparing an engineered immune cell of any one of claims 1-33, wherein the kit comprises a container, and within the container:

63. The kit of claim 62, wherein the first and second nucleic acid sequences are independent or linked.

64. The kit of claim 62, wherein the first and second nucleic acid sequences are in the same or different containers; or the first and second nucleic acid sequences are on the same or different vectors.

65. The kit of claim 62, further comprising a third nucleic acid sequence between the first and second nucleic acid sequences when the first and second nucleic acid sequences are in the same vector, the third nucleic acid sequence comprising a third expression cassette for expressing a connecting peptide.

66. The kit of claim 65, wherein the linker peptide is P2A, F a or T2A.

67. The kit of claim 65, wherein the linker peptide comprises a cleavage site for Furin protease and/or a linker short peptide upstream thereof.

68. The kit of claim 62, further comprising a 5' untranslated region (5 ' utr) element of GPX4 upstream of the second expression cassette and/or a 3' utr element of GPX4 downstream of the second expression cassette in the second nucleic acid sequence.

69. The kit of claim 68, wherein the nucleotide sequence of the 5' utr element is set forth in SEQ ID No. 7.

70. The kit of claim 68, wherein the nucleotide sequence of the 3' utr element is set forth in SEQ ID No. 8.

71. The kit of claim 64, wherein the vector is a viral vector.

72. The kit of claim 71, wherein the viral vector comprises the first and second nucleic acid sequences in tandem.