WO2019109980A1 - Chimeric protein, and immune effector cell expressing same and application thereof - Google Patents

Abstract

Provided are an immune effector cell expressing a chimeric protein and an application thereof. The immune effector cell expresses a chimeric protein containing an extracellular domain of a TGF-β receptor and an intracellular signal domain of a receptor of an IL-2 family protein. Further provided are a pharmaceutical composition containing the immune effector cell and a use of the immune effector cell or the pharmaceutical composition for the preparation of a medicament for preventing or treating tumors or pathogenic microorganism infection. The immune effector cell expressing the chimeric protein of the present invention is capable of transforming stimulation of TGF-β into a positive signal of IL-2, IL-7, or IL-21, and inhibiting TGF-β-induced Treg differentiation, thereby promoting survival and proliferation of immune cells, and is also capable of reducing the effect of TGF-β on cell killing ability.

Description

Chimeric protein, immune effector cell expressing chimeric protein and application thereof Technical field

The present invention belongs to the field of adoptive cell therapy; in particular, the present invention relates to a chimeric protein and a genetically engineered immune effector cell, which can significantly enhance the proliferation and survival of immune cells and enhance their cell killing ability. .

Background technique

In recent years, adoptive cell therapy such as CAR-T therapy and TCR-T therapy have shown good results in the field of hematoma, but the treatment of solid tumors is not good, because cancer cells in solid tumors can be around them. A tumor microenvironment is formed to support the growth and metastasis of cancer cells. Immunosuppressive cytokines such as IL-4, IL-10, TGF-β, etc., which are abundantly expressed in the tumor microenvironment, inhibit the antitumor activity of CAR-T cells. Moreover, the presence of TGF-β also induces T cell differentiation into regulatory T cells (Treg), and regulatory T cells further inhibit T cell killing.

Some researchers have tried to suppress the TGF-β signal to block the inhibition of T cells by TGF-β. For example, Foster et al. ("Antitumor Activity of EBV-specific T Lymphocytes Transduced With a Dominant Negative TGF-β Receptor", J Immunother, 2008) reported transfection of a dominant negative TGF-β receptor to block TGF-β signaling. Transduction, but this method only blocks the inhibition of T cells by TGF-β. Wang et al. ("Augmented anti-tumor activity of NK-92 cells expressing chimeric receptors of TGF-βR II and NKG2D", Cancer Immunol Immunother, 2017) reported the extracellular domain of TGF-β receptor II and the intracellular domain of NKG2D. Chimeric, the chimeric receptor can enhance the secretion of IFNγ by NK-92 cells and kill the target cells, but the therapeutic effect is not enhanced.

The lack of immune effector cells capable of effectively promoting the proliferation and survival of immune cells in the prior art is a subject that has been studied in the field for a long time.

Summary of the invention

The object of the present invention is to significantly enhance the ability of immune cells to proliferate and survive, and to enhance their cell killing ability, and the object of the present invention is achieved by a specific chimeric protein and genetically engineered immune effector cells.

In a first aspect, the invention provides an immune effector cell expressing a chimeric protein comprising an extracellular domain of a TGF-beta receptor and an intracellular signal domain of a receptor for an IL-2 family protein.

In a specific embodiment, the immune effector cell further expresses a chimeric receptor capable of recognizing a target antigen; preferably, the chimeric receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR) a T cell fusion protein (TFP), or a T cell antigen coupler (TAC); more preferably, the chimeric receptor is a chimeric antigen receptor (CAR).

In a specific embodiment, the extracellular domain of the TGF-beta receptor comprises an extracellular domain of TGF-beta receptor I and/or an extracellular domain of TGF-beta receptor II; preferably a cell of TGF-beta receptor II Outland.

In a specific embodiment, the receptor intracellular signal domain of the IL-2 family protein is selected from the group consisting of an IL-2 receptor, an IL-4 receptor, an IL-7 receptor, an IL-9 receptor, and an IL- An intracellular signal domain of any of the 15 receptors, IL-21 receptors, or a combination of at least two;

Preferably, an intracellular signal domain selected from any of the IL-2 receptor, the IL-7 receptor, and the IL-21 receptor;

More preferably, the receptor intracellular signal domain of the IL-2 family protein is the intracellular signal domain of the IL-7 receptor or the intracellular signal domain of the IL-21 receptor.

In a specific embodiment, the receptor intracellular signal domain of the IL-2 family protein comprises an intracellular domain of IL-2RG.

In a specific embodiment, the receptor intracellular signal domain of the IL-2 family protein comprises an intracellular domain of IL-2Rβ, IL-7R or IL-21R.

In a specific embodiment, the receptor intracellular domain of the IL-2 family protein comprises the intracellular domain of IL-2RG, and the intracellular domain of IL-2Rβ, IL-7R, or IL-21R.

In a specific embodiment, the chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular signal domain of the alpha subunit of a receptor of any of the IL-2 family proteins;

Preferably, the alpha subunit of the receptor of the IL-2 family protein is selected from the group consisting of IL-2Rβ, IL-7R, or IL-21R; more preferably IL-7R or IL-21R.

In a specific embodiment, the extracellular domain and the intracellular signal domain have a transmembrane domain; preferably, the transmembrane domain is a transmembrane domain of a receptor for an IL-2 family protein.

In a specific embodiment, the chimeric protein comprises a first chimeric protein and a second chimeric protein,

The first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein ;with

The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein .

In a specific embodiment, the first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular signal domain of IL-2RG;

The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular signal domain of the alpha subunit of the receptor of the IL-2 family protein.

In a specific embodiment, the intracellular signal domain of the alpha subunit of the receptor for the IL-2 family protein is selected from the intracellular signal domain of IL-2Rβ, IL-7R, or IL-21R.

In a specific embodiment, the extracellular domain of the TGF-beta receptor and the intracellular signal domain of the receptor of the IL-2 family protein are wild-type or mutant.

In a specific embodiment, the amino acid sequence of the extracellular domain of TGF-beta receptor I has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:4;

The amino acid sequence of the extracellular domain of TGF-beta receptor II has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 12;

The amino acid sequence of the intracellular domain of IL-2RG has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 8.

In a specific embodiment, the amino acid sequence of the intracellular domain of IL-2Rβ has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 16;

The amino acid sequence of the intracellular domain of IL-7R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:20;

The amino acid sequence of the intracellular domain of IL-21R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 24.

In a specific embodiment, the amino acid sequence of the first chimeric protein has at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 27; the amino acid sequence of the second chimeric protein is The amino acid sequence set forth in any one of SEQ ID NO: 28, 29, or 30 has at least 90% homology.

In a specific embodiment, the immune effector cell is selected from any one of T cells, natural killer cells, natural killer T cells, DNT cells, mast cells, or bone marrow-derived phagocytic cells, or a combination thereof; preferably The immune effector cells are selected from the group consisting of T cells.

In a specific embodiment, the target antigen is a tumor antigen or a pathogenic microorganism antigen.

In a preferred embodiment, the target antigen is a pathogenic microorganism antigen, the pathogenic microorganism including a virus, a bacterium, a fungus, a protozoa or a parasite; more preferably, the pathogenic microorganism is a virus; or more preferably, The pathogenic microorganism is selected from the group consisting of a cytomegalovirus, an Epstein-Barr virus, a human immunodeficiency virus, and an influenza virus;

In a preferred embodiment, the target antigen is a tumor antigen, and preferably, the tumor antigen comprises:

Prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, MART-1, gp100, tyrosinase, WT- I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, CLDN18.2, folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2 , 5T4, 8H9, αvβ6 integrin, B cell mature antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, kappa light chain, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2 IL11Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, fibronectin, tenascin or More preferably, the tumor antigen is phosphatidylinositol 3.

In a specific embodiment, the target antigen is an antigen of a solid tumor.

In a specific embodiment, the amino acid sequence of the extracellular antigen recognition domain of the chimeric antigen receptor has at least 90% homology to the sequence set forth in any one of SEQ ID NOs: 31-35.

In a second aspect, the present invention provides a pharmaceutical composition comprising the immune effector cell of the first aspect.

In a third aspect, the present invention provides the use of the immune effector cell of the first aspect or the pharmaceutical composition of the second aspect for the preparation of a medicament for preventing or treating a tumor or a pathogenic microorganism infection.

In a fourth aspect, the invention provides a chimeric protein comprising an extracellular domain of a TGF-beta receptor and an intracellular signal domain of a receptor for an IL-2 family protein.

In a preferred embodiment, the extracellular domain of the TGF-beta receptor comprises an extracellular domain of TGF-beta receptor I and/or an extracellular domain of TGF-beta receptor II, preferably a cell of TGF-beta receptor II Outland;

In a preferred embodiment, the receptor intracellular signal domain of the IL-2 family protein is selected from the group consisting of an IL-2 receptor, an IL-4 receptor, an IL-7 receptor, an IL-9 receptor, and an IL- An intracellular signal domain of at least one of the 15 receptor, IL-21 receptor or a combination of at least two; preferably, selected from the group consisting of an IL-2 receptor, an IL-7 receptor, and an IL-21 receptor The intracellular signal domain of any of the receptors; more preferably, the receptor intracellular signal domain of the IL-2 family protein is the intracellular signal domain of the IL-7 receptor or the cell of the IL-21 receptor Internal signal domain.

In a specific embodiment, the chimeric protein comprises the extracellular domain of TGF-beta receptor II and the intracellular signal domain of the alpha subunit of the receptor of any of the IL-2 family proteins.

In a preferred embodiment, the alpha subunit of the receptor for the IL-2 family protein is selected from the group consisting of IL-2Rβ, IL-7R, or IL-21R; preferably, IL-7R or IL-21R.

In a specific embodiment, the chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular signal domain of IL-2RG.

In a specific embodiment, the extracellular domain of the TGF-beta receptor and the intracellular signal domain of the receptor of the IL-2 family protein are wild-type or mutant.

In a specific embodiment, the amino acid sequence of the extracellular domain of TGF-beta receptor I has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:4;

The amino acid sequence of the extracellular domain of TGF-beta receptor II has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:12.

In a specific embodiment, the amino acid sequence of the intracellular domain of IL-2RG has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:8.

In a specific embodiment, the amino acid sequence of the intracellular domain of IL-2Rβ has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 16;

The amino acid sequence of the intracellular domain of IL-7R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:20;

The amino acid sequence of the intracellular domain of IL-21R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 24.

In a specific embodiment, the extracellular domain and the intracellular signal domain have a transmembrane domain;

Preferably, the transmembrane domain is a transmembrane domain of a receptor for an IL-2 family protein.

In a specific embodiment, the amino acid sequence of the chimeric protein has at least 90% homology to the amino acid sequence set forth in any one of SEQ ID NO: 27, 28, 29, or 30;

Preferably, the amino acid sequence of the chimeric protein is as set forth in any one of SEQ ID NOs: 27, 28, 29, or 30.

In a specific embodiment, the chimeric protein comprises a first chimeric protein and a second chimeric protein,

The first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein ;with

The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein .

In a preferred embodiment, the first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular signal domain of IL-2RG;

The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular signal domain of the alpha subunit of the receptor of the IL-2 family protein.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 is a schematic diagram showing the structure of a chimeric protein.

Figure 2A is a plasmid map of plasmid 1 expressing the first chimeric protein and IL-2Rβ, Figure 2B is a plasmid map of plasmid 2 expressing the first chimeric protein and IL-7RA, and Figure 2C is the expression of the first chimeric protein and Plasmid map of plasmid 3 of IL-21R.

Figure 3 shows the results of Western blot analysis of STAT3/5 phosphorylation levels.

Figure 4A is a Foxp3 flow pattern in the CD4+CD25+ population and Figure 4B is a histogram representation of the Foxp3+ population ratio.

Figure 5 shows the sustained killing ability of chTR7/21 cells against tumor cells under TGF-β stimulation.

Figure 6 is a flow diagram of the preparation of Huh7-TGFβ.

Figure 7 shows the results of in vitro killing when the target cells are Huh7 and SK-hep1-GPC3 cells.

Figure 8 shows the in vitro killing results when the target cells are Huh7-TGFβ and SK-hep1-GPC3-TGFβ.

Detailed ways

After extensive and intensive research, the inventors found that the chimeric protein containing the extracellular domain of the TGF-β receptor and the intracellular signal domain of the receptor of the IL-2 family protein and the receptor recognizing the target antigen are co-expressed in the immune effect. On the basis of the present invention, the present invention can be completed not only by inhibiting TGF-β in the tumor microenvironment but also by increasing the proliferation and survival levels of immune cells.

To facilitate an understanding of the technical solutions provided herein, the definitions of methods and/or terms herein are described below.

the term

The term "immune effector cells (also known as immune cells)" refers to cells involved in an immune response, for example, to promote an immune effector response. Examples of immune effector cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, DNT cells, mast cells, and bone marrow-derived phagocytic cells. For example, the T cells may be T cells directly derived from peripheral blood, or may be subtypes of T cells, such as CD8+ T cells, CD4+ T cells, α/β T cells, γ/δT cells, and the like.

The term "chimeric antigen receptor" or "CAR" refers to a group of polypeptides which, when administered in an immune effector cell, confer specificity to a target cell target antigen (eg, a tumor antigen) and have cells. The internal signal is generated. CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain (also known as a transmembrane domain), and a cytoplasmic signaling domain (also referred to herein as an "intracellular domain").

The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell for regulating cells via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. Activity.

The term "T cell receptor (TCR)", a characteristic marker on the surface of all T cells, binds to CD3 with a non-covalent bond to form a TCR-CD3 complex. The role of TCR is to recognize antigens. TCR is a heterodimer composed of two different peptide chains, consisting of two peptide chains, α and β. Each peptide chain can be further divided into variable region (V region), constant region (C region), transmembrane. The region and the cytoplasmic region are several parts; it is characterized by a short cytoplasmic region. The TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (Vα, Vβ) has three hypervariable regions CDR1, CDR2, and CDR3, among which the CDR3 mutation is the largest, which directly determines the TCR antigen. Binding specificity. When the TCR recognizes the MHC-antigen peptide complex, CDR1, CDR2 recognizes and binds to the side wall of the MHC molecule antigen binding groove, and CDR3 binds directly to the antigen peptide. TCR is divided into two categories: TCR1 and TCR2; TCR1 consists of two chains of γ and δ, and TCR2 consists of two chains of α and β.

The term "T Cell Fusion Protein (TFP)", includes various polypeptide-derived recombinant polypeptides that constitute a TCR, which are capable of i) binding to a surface antigen on a target cell, and ii) complex TCR complexes The interaction of other polypeptides is usually localized on the surface of T cells. TFP consists of an antigen binding domain consisting of a TCR subunit and a human or humanized antibody domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular domain. a stimulating domain of an internal signal domain; the TCR subunit and the antibody domain are operably linked, wherein the extracellular, transmembrane, intracellular signal domain of the TCR subunit is derived from CD3 epsilon or CD3 gamma, and the TFP is integrated TCR expressed on T cells.

By "effective link" is meant a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of a heterologous nucleic acid sequence. For example, when the first nucleic acid sequence is located in a functional regulatory region of the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter is required to affect transcription and expression of a coding sequence, then this promoter can be operably linked to the coding sequence.

The term "T Cell Antigen Coupler (TAC)" includes three functional domains: a tumor targeting domain, including a single chain antibody, a designed ankyrin repeat protein (DARPin). Or other targeting group; 2 extracellular domain domain, is a single-chain antibody that binds to CD3, such that the TAC receptor is adjacent to the TCR receptor; 3 the transmembrane region and the intracellular region of the CD4 co-receptor, wherein The intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.

The term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.

The term "tumor microenvironment" refers to any and all elements of the tumor environment, including elements that create a structural and/or functional environment for a malignant process to survive and/or expand and/or spread.

The terms "TGF-β" and "TGFb" have the same meaning herein, and the term "extracellular domain of TGF-beta receptor" includes the extracellular domain of the native TGF-beta receptor and the extracellular domain of the mutant TGF-beta receptor. . As used herein, the sequence of the extracellular domain of a "native" or "wild-type" TGF-beta receptor primarily refers to the extracellular domain sequence of a human TGF-beta receptor, whether purified from a natural source or using recombinant techniques.

It will be understood that the extracellular domain of the TGF-beta receptor according to the present disclosure comprises a fragment that may be shorter or longer than the extracellular domain protein of the native TGF-beta receptor, as long as the extracellular domain protein fragment of the TGF-beta receptor remains The ability to bind TGF-β. It is also to be understood that the disclosure encompasses nucleic acid molecules encoding TGF-[beta] receptors described herein or known in the art, including, but not limited to, RNA sequences corresponding to the DNA sequences described herein.

The signal transduction pattern of TGF-β is bound by TGF-β receptor II (also referred to herein as TGFbRII) to TGF-β, and then recruits TGF-β receptor I (also referred to herein as TGFbRI) to initiate intracellular Recruitment and phosphorylation of Smad2/3.

The term "IL-2 family protein" includes IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, etc., and the "receptor of IL-2 family protein" contains γ. The chain (IL-2RG, also referred to herein as IL2RG, whose sequence is set forth in SEQ ID NO: 43) acts as a cytokine for its receptor subunit, and is also shared by each specific Rα (eg, IL-2 and IL-15). IL-2Rβ (also referred to herein as IL2RB), IL-7R (also referred to herein as IL7R), IL-21R (also referred to herein as IL21R), and the shared gamma chain subunit (IL-2RG) Rα recognizes extracellular cytokines, recruits IL-2RG to bind and initiates intracellular phosphorylation of STAT1/3/4/5.

The term "antigen" or "Ag" refers to a molecule that can provoke an immune response. The immune response can involve the production of antibodies, or activation of specific immunocompetent cells, or both. It will be apparent to those skilled in the art that any macromolecule, including substantially all proteins or peptides, can serve as an antigen. Furthermore, the antigen may be derived from recombinant DNA or genomic DNA. It will be apparent to those skilled in the art that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that can elicit an immune response, thus encoding an "antigen" (as the term is used herein). Furthermore, it will be apparent to those skilled in the art that the antigen need not be encoded only by the full length nucleotide sequence of the gene. It will be readily apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, which may be arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, it will be apparent to those skilled in the art that the antigen need not be encoded by a "gene". It will be readily apparent that the antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. The biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell or a liquid, and other biological components.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

The term "llow virus" refers to the genus of the family Lentiviridae. Lentiviruses are unique in retroviruses and are capable of infecting non-dividing cells; they are capable of delivering a significant amount of genetic information into the DNA of a host cell, whereby they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses.

The term "homology" or "similarity" refers to between two polymer molecules, for example between two nucleic acid molecules, for example between two DNA molecules or two RNA molecules, or between two polypeptide molecules. Unit sequence identity. When a subunit position is occupied by the same monomer subunit in two molecules, for example when two DNA molecules are occupied by adenosine at one position, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matched or homologous positions; for example, when half of the positions in the two sequences (for example, 5 positions in a polymer of 10 subunits) are homologous The two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary test cells and their progeny.

Chimeric protein of the invention

The present invention provides a chimeric protein comprising an extracellular domain of a TGF-beta receptor and an intracellular signal domain of a receptor for an IL-2 family protein. Co-expression of this chimeric protein and a receptor recognizing the target antigen on immune effector cells can inhibit TGF-β in the tumor microenvironment and increase the proliferation and survival of immune cells.

In a specific embodiment, the extracellular domain of the TGF-beta receptor is the extracellular domain of TGF-beta receptor I and/or the extracellular domain of TGF-beta receptor II. In another specific embodiment, the receptor intracellular signal domain of the IL-2 family protein is selected from the group consisting of an IL-2 receptor, an IL-4 receptor, an IL-7 receptor, an IL-9 receptor, An intracellular signal domain of any of the IL-15 receptor, IL-21 receptor, or a combination of at least two; preferably, selected from the group consisting of an IL-2 receptor, an IL-7 receptor, and an IL-21 The intracellular signal domain of any of the receptors; more preferably, the receptor intracellular signal domain of the IL-2 family protein is the intracellular signal domain of the IL-7 receptor or the IL-21 receptor Intracellular signal domain.

In a further preferred embodiment, the extracellular domain of the TGF-beta receptor is preferably the extracellular domain of TGF-beta receptor II, and the receptor intracellular signal domain of the IL-2 family protein is the IL-2 family. The intracellular signal domain of the alpha subunit of the protein receptor. In a specific embodiment, the alpha subunit of the receptor for the IL-2 family protein is selected from the group consisting of IL-2Rβ, IL-7R, or IL-21R; preferably IL-7R or IL-21R.

It is known to those skilled in the art that polypeptides having certain amino acid sequence identity or homology to the extracellular domain or intracellular domain described above may also have the same or similar functions. Thus, in some embodiments, the extracellular domain of TGF-beta receptor I, the extracellular domain of said TGF-beta receptor II, the intracellular domain of said IL-2RG, said IL- The amino acid sequence of the intracellular domain of 2Rβ, the intracellular domain of IL-7R, and the intracellular domain of IL-21R is SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 8, respectively. The amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or 99% homology; and the amino acid sequence of the chimeric protein of the invention has at least 90%, 91%, 92%, 93 with the amino acid sequence set forth in any one of SEQ ID NO: 27, 28, 29, or %, 94%, 95%, 96%, 97%, 98% or 99% homology.

The chimeric proteins of the invention also have a transmembrane domain between the extracellular domain and the intracellular signal domain. The "transmembrane domain" may include a transmembrane domain of a plurality of natural receptor proteins, as long as it functions to connect the extracellular and intracellular regions of the receptor and anchor to the cell membrane, and thus, the incorporation of the present invention The transmembrane domain in the protein is not restricted to any particular transmembrane domain, including but not limited to the TGF-beta receptor, the transmembrane region of the receptor of the IL-2 family protein; preferably, the transmembrane domain is The transmembrane domain of the receptor for the IL-2 family of proteins.

Further, the inventors have found that when the chimeric protein of the present invention comprises the first chimeric protein and the second chimeric protein, it can have more excellent technical effects. In a specific embodiment, the first chimeric protein comprises an extracellular domain of TGF-beta receptor I and a cell selected from the intracellular signal domain of IL-2RG or the alpha subunit of the receptor of the IL-2 family protein An intracellular domain of an internal signal domain; said second chimeric protein comprising an extracellular domain of TGF-beta receptor II and an alpha subunit selected from the group consisting of an intracellular signal domain of IL-2RG or a receptor of an IL-2 family protein The intracellular domain of the intracellular signal domain. For example, the first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular domain of IL-2RG; the second chimeric protein comprises an extracellular domain of TGF-beta receptor II and IL-2 The intracellular signal domain of the alpha subunit of the receptor of the family protein.

Chimeric antigen receptor

"Chimeric Antigen Receptor (CAR)" refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that activates T cells. Typically, the extracellular binding domain of CAR is derived from a mouse or humanized or human monoclonal antibody.

Chimeric antigen receptors typically comprise a (fine) extracellular antigen binding region. In some embodiments, the extracellular antigen binding region can be fully human. In other cases, the extracellular antigen binding region can be humanized. In other instances, the extracellular antigen binding region can be murine or the chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region can be non-human.

In some cases, the extracellular antigen binding region comprises a hinge or spacer. The terms hinge and spacer are used interchangeably. The hinge can be considered as part of a CAR for providing flexibility to the extracellular antigen binding region. In some cases, the hinge can be used to detect CAR on the cell surface of a cell, particularly when detecting antibodies to the extracellular antigen binding region are ineffective or available. For example, the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the location of the extracellular antigen binding region that targets the epitope on the target.

The transmembrane domain of CAR can anchor the CAR to the plasma membrane of the cell, such as the transmembrane domain of CD8, the transmembrane domain of CD28, and the like. The skilled person can replace it according to the known transmembrane domain.

The intracellular signal domain of CAR may be responsible for activating at least one of the effector functions of the immune response cells into which the CAR has been placed. CAR can induce effector functions of T cells, for example, the effector function is cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signal domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling region can generally be used, in many cases it is not necessary to use the entire chain of the signal domain. In some cases, a truncated portion of the intracellular signaling region is used. In some instances, the term "intracellular signal domain" includes any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.

The intracellular signaling domain of CAR can be selected from any of the domains of Table 1. The intracellular signaling region of CAR may further comprise one or more costimulatory domains. The intracellular signaling region may comprise a single costimulatory domain, such as an ζ chain (first generation CAR) or it is with CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling region can comprise two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation). In some cases, signals generated by the CAR may be combined with an auxiliary or costimulatory signal. For costimulatory signaling domains, chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains. Several receptors have been reported to provide co-stimulation for T cell activation including, but not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB. The signaling pathways used by these costimulatory molecules work synergistically with the primary T cell receptor activation signal. The signals provided by these costimulatory signaling regions can act synergistically with primary effect activation signals derived from one or more ITAM motifs (eg, the CD3zeta signal transduction domain) and can fulfill the requirements for T cell activation.

Table 1. Costimulatory domains

analysis

Chimeric proteins or immune effector cells expressing chimeric proteins can be analyzed using standard methods known in the art or described herein.

application

Immune effector cells comprising a chimeric protein as described herein and a receptor that recognizes a target antigen can be used in a variety of therapeutic applications. In general, the immune effector cells described herein can be used in the treatment or prevention of any disease, disorder, or condition involving a cell that expresses TGF-β and that would benefit from inhibition of cell proliferation or promotion of cell death. In some embodiments, it can be used to induce apoptosis or cell death, or to treat disorders associated with abnormal apoptosis or cell proliferation, such as cancer.

The term "cancer," "cancer," "hyperproliferation," or "tumor," as used herein, refers to a cell that has the ability to grow automatically (eg, an abnormal state or condition characterized by a proliferation of cells that are proliferating). Hyperproliferative or neoplastic disease states can be classified as pathological types (eg, because they deviate from normal but are not associated with disease states). Thus, "cancer" or "tumor" refers to any unwanted growth of a cell that has no physiological function. The term cancer includes cell growth that is technically benign but may present a risk of becoming malignant. "Malignant" refers to the abnormal growth of any cell type or tissue. The term "malignant" includes cell growth that is technically benign but at risk of becoming malignant. The term also includes any cancer, cancer, neoplasm, tumor formation or tumor. Thus, these terms are meant to include all types of cancer growth or tumorigenesis processes, metastatic tissues or malignant transformed cells, tissues or organs, whether of histopathological type or invasive stage.

Most cancers are divided into three large histological categories: cancer, which is the majority of cancers and epidermal cells or covers organs, glands, or other body structures (eg, skin, uterus, lung cancer, breast cancer, prostate cancer). , cancer of the outer or inner surface of the stomach, intestines, and often metastasis; sarcoma, which is derived from connective tissue or supporting tissue (eg, bone, cartilage, tendons, ligaments, fat, muscle); and blood Tumors, which are derived from bone marrow and lymphoid tissues. Examples of cancer include, but are not limited to, cancer, sarcoma, and hematological tumor formation disorders such as leukemia.

The cancer can be an adenocarcinoma (which is typically in an organ or gland that can be secreted, such as the breast, lung, colon, prostate, or bladder), or can be a squamous cell carcinoma (which is derived from the squamous epithelium and is generally large in the body). Part of the area is formed).

Sarcoma can be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), striated muscle (skeletal muscle), mesothelioma or mesothelioma (membranous lining of body cavity), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelial blood vessels), liposarcoma (fat), glioma or astrocytoma (found in the brain's neurogenic connective tissue), mucinous sarcoma (primary embryonic connective tissue) or between Leaf cell tumor or mesodermal mixed tumor (mixed connective tissue type).

Hematopoietic tumor-forming disorders include proliferative/neoplastic cells involved in the origin of hematopoiesis, for example, derived from the myeloid, lymphoid or erythroid cell lines or their precursor cells. Preferably, the disease is derived from poorly differentiated acute leukemia (eg, erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myelogenous leukemia (CML); lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-line acute lymphoblastic leukemia and T-line acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia, and Waldens Waldenstrom's macroglobulinemia.

Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymph Cellular leukemia (LGF), Hodgkin's disease, and Ris-Sick disease.

These cancers can also be named according to the organ from which the cancer is derived, ie, the "primary site", such as the breast, brain, lung, liver, skin, prostate, testis, bladder, colon and rectum, cervix, uterus, and the like. This name is adhered to even if the cancer is transferred to another part of the body that is different from the original site. Cancers named according to the primary site can be associated with histological classification. For example, lung cancer is generally small cell lung cancer and non-small cell lung cancer, which may be squamous cell carcinoma, adenocarcinoma, large cell carcinoma; skin cancer is usually basal cell carcinoma, squamous cell carcinoma or melanoma. Lymphoma may occur in lymph nodes associated with the head, neck, and chest as well as in abdominal lymph nodes or axillary or inguinal lymph nodes.

"Solid tumor" refers to tumors and/or metastases other than lymphoma (wherever they are), such as the brain or other central nervous system tumors (eg meningioma, brain tumor, myeloma, cranial neuroma, and central nervous system) Other parts of the tumor, such as malignant glioma or medulloblastoma; head and / or neck cancer; breast tumors; circulatory tumors (such as the heart, mediastinum and pleura, and other organs in the thoracic tumor, hemangiomas and Tumors associated with vascular tissue; excretory system tumors (eg, kidney, renal pelvis, ureter, bladder, other and urinary organs not specifically indicated); gastrointestinal tumors (eg, esophagus, stomach, small intestine, colon, colorectum, recto sigmoid colon) Combination point, rectum, anus, anal canal), other liver and intrahepatic bile duct, gallbladder, biliary tract and other parts of the pancreas and other digestive organs; head and neck; mouth (lips, tongue, gums, Bottom of the mouth, other parts of the mouth and mouth, other parts of the parotid and salivary glands, tonsils, oropharynx, nasopharynx, pear sinus, lower pharynx and lips, other parts of the mouth and pharynx) Reproductive system tumors (eg vulva, vagina, cervix, uterus, uterus, ovary and other parts of the female reproductive organs, placenta, penis, prostate, testes and other parts of the male reproductive organs); respiratory tumors (eg nasal and middle ear) , paranasal sinus, larynx, trachea, bronchus and lungs, such as small cell lung cancer or non-small cell lung cancer); skeletal system tumors (such as bone and articular cartilage of the extremities, osteoarticular cartilage and other parts); skin tumors (such as skin malignant melanin) Tumor, non-melanoma skin cancer, cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma, mesothelioma, Kaposi's sarcoma; and other tissues, including peripheral and autonomic nervous systems, connective and soft tissues, retroperitoneal cavity and peritoneum , Eyes and attachments, thyroid, adrenal and other endocrine glands and related structural tumors, secondary and unspecified malignant lymphomas, secondary malignancies of the respiratory and digestive system, and secondary malignancies elsewhere.

When referring to tumors, tumor diseases, cancers or malignant tumors above and below, metastasis of the primary organ or tissue and/or any other site is also indicated, either alone or simultaneously, regardless of where the tumor and/or metastases are located. .

Pharmaceutical composition

Pharmaceutical compositions in accordance with the present disclosure may comprise the immune effector cells provided herein and one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients, and adjuvants. These compositions may be suitable for use in the treatment of the therapeutic indications described herein.

Other active ingredients may be included in the composition if desired. Thus, in some embodiments, the immune effector cells can be administered in a therapeutically effective amount to treat one or more cancers or in combination with other therapies. The immune effector cells can be administered before, during or after treatment with an anti-tumor or other therapy. An "anti-cancer treatment" is a compound, composition or treatment (eg, surgery) that prevents or delays the growth and/or metastasis of cancer cells. Such anti-cancer therapies include, but are not limited to, surgery (eg, removal of all or part of a tumor), chemotherapy drug therapy, radiation, gene therapy, hormone control, immunotherapy (eg, therapeutic antibodies and cancer vaccines), and antisense or RNAi Oligonucleotide treatment. Examples of useful chemotherapeutic agents include, but are not limited to, hydroxyurea, busulphan, cisplatin, carboplatin, chlorambucil, melphalan, cyclophosphamide, ifosfamide, daunubicin ), doxorubicin, epirubicin, mitoxantrone, vincristine, vinblastine, vinorelbine, etoposide, teniposide, paclitaxel, docetaxel, gemcitabine, cytosine, arabinose Cytidine, bleomycin, neocarcinostatin, suramin, taxol, mitomycin C, avastin, fluorouracil, temozolamide, and the like. The immune effector cells are also suitable for use by standard combination therapies using two or more chemotherapeutic drugs. It should be understood that anti-cancer therapies include new compounds or treatments developed in the future.

The immune effector cells can also be used in combination with a radiation sensitizer such as a radiotherapy sensitizer. In general, a sensitizer is any agent capable of increasing the activity of the immune effector cells. For example, a sensitizer will increase the ability of the fusion protein to inhibit cancer cell growth or kill cancer cells. Exemplary sensitizers include antibodies against IL-10, bone morphogenetic proteins, and HDAC inhibitors (see, for example, Sakariassen et al., Neoplasia 9(11): 882-92, 2007).

The immune effector cells may be used as part of a neoadjuvant therapy (to primary therapy) as part of an adjuvant therapy regimen in which the goal is to cure cancer in the subject. The immune effector cells can also be administered at different stages of tumorigenesis and progression, including in advanced and/or invasive neoplasms (eg, by topical treatment in a subject (eg, a dominant disease that cannot be cured by surgery or radiotherapy) , metastatic disease. Local phase disease and/or refractory tumor (eg, treatment of cancer or tumor that does not respond to treatment) is administered at various stages. "Primary therapy" refers to the initial diagnosis of cancer in a subject. First-line treatment. Exemplary primary therapy may involve surgery, a wide range of chemotherapy, and radiation therapy. "Auxiliary therapy" refers to a therapy that follows a primary therapy and is administered at a risk of recurrence. The adjuvant systemic treatment begins soon after the primary therapy, for example at 2, 3, 4, 5, or 6 weeks after the last primary therapy treatment to delay recurrence, prolong survival or cure the subject. As discussed herein, it is contemplated that the immune effector cells can be used alone or as part of an adjuvant therapy with one or more Other chemotherapeutic drugs are used in combination. The combination of the immune effector cells and standard chemotherapeutic agents can enhance the efficacy of chemotherapy and, therefore, can be used to improve standard cancer therapy.

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

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

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

A pharmaceutical composition comprising one or more immune effector cells can be formulated as a sterile injectable aqueous or oil according to methods known in the art and using one or more suitable dispersing or wetting agents and/or suspending agents. A suspension. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., "Molecular Cloning Experimental Guide (Third Edition)" (Science Press, 2002). Or according to the conditions recommended by the manufacturer.

Example 1. Construction of a vector expressing a chimeric protein

The plasmid expressing the chimeric protein comprising the structure shown in Fig. 1 was constructed as follows.

(1) Human TGF-β receptor I signal peptide (SEQ ID NO: 2), extracellular domain of TGF-β receptor I (SEQ ID NO: 4) and human IL-2RG transmembrane region (SEQ ID NO: 6) The nucleotide sequence of the coding sequence of the IL-2RG intracellular domain (SEQ ID NO: 8) is ligated to construct the nucleotide sequence of the first chimeric protein chIL2RG (SEQ ID NO: 37, which encodes the amino acid sequence as SEQ. ID NO: 27)).

(2) Human TGF-β receptor II signal peptide (SEQ ID NO: 10), TGF-β receptor II extracellular domain (SEQ ID NO: 12) and human IL-2Rβ transmembrane region (SEQ ID NO: 14) , the nucleotide sequence encoding the intracellular region of IL-2Rβ (SEQ ID NO: 16) is ligated to construct the nucleotide sequence of the second chimeric protein TGFβRII-IL2Rβ (SEQ ID NO: 38, the encoded amino acid sequence thereof SEQ ID NO: 28).

(3) Human TGF-β receptor II signal peptide (SEQ ID NO: 10), TGF-β receptor II extracellular domain (SEQ ID NO: 12) and human IL-7RA transmembrane region (SEQ ID NO: 18) , the coding sequence of the intracellular region of IL-7RA (SEQ ID NO: 20) is ligated to construct the nucleotide sequence of the second chimeric protein TGFβRII-IL7RA (SEQ ID NO: 39, the encoded amino acid sequence is SEQ ID NO :29)).

(4) Human TGF-β receptor II signal peptide (SEQ ID NO: 10), TGF-β receptor II extracellular domain (SEQ ID NO: 12) and human IL-21R transmembrane region (SEQ ID NO: 22) , the coding sequence of the IL-21R intracellular domain (SEQ ID NO: 24) is ligated to construct the nucleotide sequence of the second chimeric protein TGFβRII-IL21R (SEQ ID NO: 40, the encoded amino acid sequence is SEQ ID NO :30)).

(5) The above first chimeric protein chIL2RG (SEQ ID NO: 27) and the second chimeric protein TGFβRII-IL2Rβ (SEQ ID NO: 28) and the second chimeric protein TGFβRII-IL7RA (SEQ ID NO: 29) The second chimeric protein TGFβRII-IL21R (SEQ ID NO: 30) was ligated with the coding sequence of the cleavage peptide F2A (SEQ ID NO: 26) and inserted into the pWPT lentiviral expression vector (purchased from Addgene). Plasmid 1 expressing the first chimeric protein and IL-2RB (the plasmid map is shown in Figure 2A), plasmid 2 expressing the first chimeric protein and IL-7RA (the plasmid map is shown in Figure 2B), and the expression A chimeric protein and plasmid 3 of IL-21R (plasmid pattern is shown in Figure 2C).

Example 2. Determination of T cell phosphorylation levels

The constructed lentiviral plasmid 1, lentiviral plasmid 2, lentiviral plasmid 3 were co-transfected into the HEK-293T cells with the lentiviral packaging plasmid, respectively, and the corresponding lentiviruses were prepared. The following were recorded as lentivirus 1 and lentivirus 2, respectively. Lentivirus 3.

Human PBMC were cultured in AIM-V medium, 2% human AB type serum was added, 500 U/mL recombinant human IL-2 was added, and CD3/CD28 antibody was added to activate magnetic beads for 48 h to obtain activated T cells. After T cells were activated, they were infected with lentivirus 1, lentivirus 2, and lentivirus 3, respectively, to obtain chimeric T cells chTR2, chTR7, and chTR21.

Serum was allowed to rest for 24 h, and blank T cell UTD (untransfected T cells, Untransfected) was used as a blank control. UTD, chTR2, chTR7, and chTR21 cells were used to divide into TGF-β1 positive group (+) and negative group (-). The culture was carried out, and TGF-β1 stimulation was not used in the negative group culture, and the recombinant group was stimulated with recombinant human TGF-β1 (5 ng/mL) for 30 min. Cell extracts were collected for Western blot analysis to analyze changes in STAT3/5 phosphorylation levels.

Western blot results are shown in Figure 3. Among them, Y705 and Y694 are tyrosine phosphorylation sites on STAT3 and STAT5, respectively. The phosphorylation level of this phosphorylation site indicates that STAT3/5 is activated. Compared with the uninfected group (UTD), the STAT3/5 phosphorylation level of chTR2/7/21 was significantly up-regulated under TGF-β1 stimulation, and was consistent with the patterns of IL-2, IL-7 and IL-21 signaling. This chimeric receptor is capable of transforming TGF-β1 stimulation into a signal of IL-2, IL-7 or IL-21. This experiment demonstrates the effectiveness of the three chimeric receptor functions provided at the molecular level.

Example 3. Effect of chimeric protein-expressing T cells on the level of Treg differentiation stimulated by recombinant human TGF-β1

Human PBMC were cultured in AIM-V medium, 2% human AB type serum and 500 U/mL recombinant human IL-2 were added to obtain the corresponding cell culture medium. The T cells chTR7, chTR21 and empty control T cell UTD obtained in Example 2 were cultured in the above cell culture medium, cultured for 4 days with recombinant human TGF-β1 (5 ng/mL), and cells were collected to label Treg markers with antibodies. CD4, CD25, and Foxp3 were subjected to flow detection, and a test group not treated with recombinant human TGF-β1 was used as a control (CTRL). As a result, as shown in Figures 4A and 4B, the ratio of Foxp3 ^{+ to} the CD4 ⁺ CD25 ⁺ population in UTD cells stimulated by TGF-β1 increased, indicating that TGF-β1 induced Treg differentiation, while the proportion of Foxp3 ^{+ in} chimeric receptor cells. Maintaining at a lower level indicates that the chimeric receptor inhibits TGF-β1-induced Treg differentiation.

Example 4. Determination of cell killing ability under TGF-β1 stimulation

In this embodiment, the selected CAR is a GPC-targeting CAR having the scFv set forth in SEQ ID NO: 31, the amino acid sequence of the CAR is set forth in SEQ ID NO: 36, and the nucleotide sequence is SEQ ID NO: 44. Shown.

Lentiviral packaging was carried out by molecular biology methods conventional in the art, and a plasmid containing the gene of CAR (SEQ ID NO: 44) was infected with 293T cells to obtain a lentivirus comprising a second generation GPC3-28z-CAR targeting GPC3. .

T cell activation: Human PBMC were cultured in AIM-V medium, 2% human AB type serum and 500 U/mL recombinant human IL-2 were added, and CD3/CD28 antibody was added to activate magnetic beads for 48 h.

T cells were activated, infected with lentivirus 2 and the second generation GPC3-28z-CAR lentivirus targeting GPC3, and T cells expressing chTR7 and CAR were obtained, which were recorded as chTR7-CAR; infected with lentivirus 3 and targeted GPC3 Generation of the LPC of GPC3-28z-CAR, T cells expressing chTR21 and CAR, designated chTR21-CAR, infected with the second generation GPC3-28z-CAR targeting GPC3 (the amino acid sequence of which is shown in SEQ ID NO: 36) The lentivirus, a T cell (GPC3-CAR-T) expressing only CAR.

The chTR7-CAR, chTR21-CAR and GPC3-CAR-T were divided into control group (CTRL) and rTGF-β1 group, CTRL group was cultured under normal conditions, and rTGF-β1 group was further added with recombinant human TGF-β1 (5 ng/mL). Treatment culture. After 4 days, the target cell was incubated with the target cell Huh7 for 48 h. After the first round of target cell killing, the collected T cells continued to be incubated with the target cell Huh7 for a second round for 48 h. The dead cells were washed with PBS, and the target cell killing was observed under the microscope. The results are shown in Fig. 5. The sustained killing ability of GPC3-CAR-T in the control group was significantly decreased under the treatment of TGF-β1, while the degree of toxicity of chTR7-CAR was decreased, and chTR21-CAR retained the cells more significantly. toxicity. These results indicate that CAR-T cells expressing chimeric receptors are more tolerant in the inhibitory environment in which TGF-β1 is present, and chimeric receptors can reduce the effect of TGF-β1 on cell killing ability. Good cytotoxicity is retained.

Example 5. Comparison of cell proliferation in vitro

The chTR7-CAR and GPC3-CAR-T prepared in Example 4 were selected for in vitro culture under different conditions:

(1) ChTR7-CAR and GPC3-CAR-T were cultured under the culture conditions of AMIV medium + 2% human AB serum, without adding IL2 and not co-incubating with tumor cells, the results showed that chTR7-CAR and GPC3-CAR There was no significant difference in proliferative capacity compared to T cells.

(2) Under the culture conditions of AIMV medium + 2% human AB type serum, chTR7-CAR and GPC3-CAR-T cells and liver cancer cell line (Huh7-TGFβ) overexpressing TGFβ1 were in a ratio of 1:3. On the fifth day, the number of chTR7-CAR has increased to 1.5 times the initial amount, while the number of GPC3-CAR-T is substantially the same as the initial amount.

Among them, Huh7-TGFβ is prepared by carrying a lentiviral expression vector plasmid pWPT-EGFP-F2A carrying TGFβ1 (the nucleotide sequence is shown in SEQ ID NO: 40, the amino acid sequence is shown as SEO ID NO: 41). -hTGFb1, Rev packaging plasmid, RRE packaging plasmid and VSV-G envelope protein pellet were mixed with transfection reagent polyethyleneimine (PEI, Polysciences) and transfected into HEK-293T cells, and transfected to obtain recombinant lentivirus solution. After concentrating the liver cancer cell line huh7 cells with the concentrated virus, the GFP-positive cells were sorted by flow to obtain Huh7-TGFβ, and the flow chart is shown in Fig. 6.

Example 6. Comparison of cell killing in vitro

Referring to the operation of Example 4, hepatoma cell lines Huh7 and SK-hep1-GPC3 cells were used as target cells, and in vitro killing of chTR7-CAR and GPC3-CAR-T under different culture conditions was performed at a target ratio of 3:1. The situation was carried out using the CytoTox96 non-radioactive cytotoxicity test kit (Promega). The specific method is described in the CytoTox 96 non-radioactive cytotoxicity test kit.

The results showed that after 18 hours of culture in RPMI-1640 medium + 10% fetal bovine serum, the killing results are shown in Figure 7. GPC3-CAR-T and chTR7 have no killing effect on SK-hep1-GPC3 and Huh7 liver cancer cells. Significant difference; after 18 hours of culture in RPMI-1640 medium + 10% fetal bovine serum + TGFβ (10 ng / ml), the killing results are shown in Figure 8, the killing effect of chTR7 on SK-hep1-GPC3 and Huh7 hepatoma cells. Both were significantly stronger than GPC3-CAR-T, indicating that the killing ability of chTR7-CAR was significantly improved in the presence of TGFβ.

Example 7. In vivo transplantation tumor experiment

A mouse subcutaneous tumor model was constructed using a liver cancer cell line (PLC/PRF/5-TGFβ1) overexpressing TGFβ1.

The preparation method of PLC/PRF/5-TGFβ1 is similar to the preparation method of Huh7-TGFβ of Example 5. The PLC/PRF/5 cell line (purchased from the Shanghai Institute of Biosciences, Chinese Academy of Sciences) was transfected with HEK-293T cells carrying the TGFβ1 gene to obtain PLC/PRF/5-TGFβ1 cells.

When chTR7-CAR and GPC3-CAR-T were given when the tumor volume was about 450 mm ² , the results showed that for over-loaded tumors, chTR7 had a better inhibitory effect on tumor growth than GPC3-CAR-T cells. After 21 days of treatment, the average tumor volume of the chTR7 group mice was about 24% smaller than that of the GPC3-CAR-T group.

In an embodiment of the invention, exemplarily, a G-cell-targeting CAR-T cell is employed, and those skilled in the art can employ CAR-T cells targeting other targets, such as targeting EGFR, in accordance with the teachings of the present application. CAR-T cells (exemplary, the sequence of the extracellular scFv of EGFR-targeting CAR-T cells is shown in SEQ ID NO: 32), such as CAR-T cells targeting CLD18A2 (exemplary, target) The sequence of the extracellular scFv to the CAR-T cells of CLD18A2 is as shown in SEQ ID NO: 33), such as CAR-T cells targeting CD19 (exemplary, extracellular to CAR-T cells targeting CD19) The sequence of the scFv is as set forth in SEQ ID NO: 34), such as a CAR-T cell targeting BCMA (exemplary, the sequence of the extracellular scFv of the BCMA-targeting CAR-T cell is set forth in SEQ ID NO: 35 ).

The sequences involved in the present invention are summarized as follows:

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (32)

An immune effector cell expressing a chimeric protein, characterized in that the chimeric protein comprises an extracellular domain of a TGF-beta receptor and an intracellular signal domain of a receptor of an IL-2 family protein. The immune effector cell according to claim 1, wherein said immune effector cell further expresses a chimeric receptor capable of recognizing a target antigen; preferably, said chimeric receptor is a chimeric antigen receptor (CAR) a T cell receptor (TCR), a T cell fusion protein (TFP), or a T cell antigen coupler (TAC); more preferably, the chimeric receptor is a chimeric antigen receptor (CAR). The immune effector cell according to claim 1 or 2, wherein the extracellular domain of the TGF-β receptor comprises an extracellular domain of TGF-β receptor I and/or an extracellular domain of TGF-β receptor II. The extracellular domain of TGF-beta receptor II is preferred. The immune effector cell according to claim 1 or 2, wherein the receptor intracellular signal domain of the IL-2 family protein is selected from the group consisting of an IL-2 receptor, an IL-4 receptor, and an IL-7 receptor. An intracellular signal domain of at least one of a body, an IL-9 receptor, an IL-15 receptor, and an IL-21 receptor, or a combination of at least two; Preferably, an intracellular signal domain selected from any of the IL-2 receptor, the IL-7 receptor, and the IL-21 receptor; More preferably, the receptor intracellular signal domain of the IL-2 family protein is the intracellular signal domain of the IL-7 receptor or the intracellular signal domain of the IL-21 receptor. The immune effector cell according to claim 4, wherein the receptor intracellular signal domain of the IL-2 family protein comprises an intracellular domain of IL-2RG. The immune effector cell according to claim 4, wherein the receptor intracellular signal domain of the IL-2 family protein comprises an intracellular domain of IL-2Rβ, IL-7R or IL-21R. The immune effector cell according to claim 4, wherein the receptor intracellular signal domain of the IL-2 family protein comprises an intracellular domain of IL-2RG, and IL-2Rβ, IL-7R, or IL. The intracellular domain of -21R. The immune effector cell according to any one of claims 1 to 7, wherein the chimeric protein comprises an extracellular domain of TGF-β receptor II and an α subunit of a receptor of any IL-2 family protein. Intracellular signal domain; Preferably, the alpha subunit of the receptor of the IL-2 family protein is selected from the group consisting of IL-2Rβ, IL-7R, or IL-21R; more preferably IL-7R or IL-21R. The immune effector cell according to claim 1 or 2, wherein the extracellular domain and the intracellular signal domain have a transmembrane domain; preferably, the transmembrane domain is an IL-2 family protein The transmembrane domain of the receptor. The immune effector cell according to any one of claims 1 to 9, wherein the chimeric protein comprises a first chimeric protein and a second chimeric protein, The first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein ;with The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein . The immune effector cell according to claim 10, wherein the first chimeric protein comprises an extracellular domain of TGF-β receptor I and an intracellular signal domain of IL-2RG; The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular signal domain of the alpha subunit of the receptor of the IL-2 family protein. The immune effector cell according to claim 11, wherein the intracellular signal domain of the α subunit of the receptor of the IL-2 family protein is selected from the group consisting of IL-2Rβ, IL-7R, or IL-21R. Intracellular signal domain. The immune effector cell according to any one of claims 1 to 12, wherein the extracellular domain of the TGF-β receptor and the intracellular signal domain of the receptor of the IL-2 family protein are wild type. Or mutant. The immune effector cell according to any one of claims 10 to 13, wherein The amino acid sequence of the extracellular domain of TGF-β receptor I has at least 90% homology with the amino acid sequence of SEQ ID NO: 4; The amino acid sequence of the extracellular domain of TGF-beta receptor II has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 12; The amino acid sequence of the intracellular domain of IL-2RG has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 8. The immune effector cell according to claim 12 or 13, wherein The amino acid sequence of the intracellular domain of IL-2Rβ has at least 90% homology with the amino acid sequence of SEQ ID NO: 16; The amino acid sequence of the intracellular domain of IL-7R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:20; The amino acid sequence of the intracellular domain of IL-21R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 24. The immune effector cell according to any one of claims 10-13, wherein the amino acid sequence of the first chimeric protein has at least 90% homology with the amino acid sequence of SEQ ID NO:27; The amino acid sequence of the second chimeric protein has at least 90% homology to the amino acid sequence set forth in any one of SEQ ID NO: 28, 29, or 30. The immune effector cell according to any one of claims 1 to 16, wherein the immune effector cell is selected from the group consisting of a T cell, a natural killer cell, a natural killer T cell, a DNT cell, a mast cell or a bone marrow-derived phagocytic cell. Any one or a combination thereof; preferably, the immune effector cells are selected from the group consisting of T cells. The immune effector cell according to claim 2, wherein the target antigen is a tumor antigen or a pathogenic microorganism antigen. The immune effector cell according to claim 18, wherein the target antigen is an antigen of a solid tumor. The immune effector cell according to claim 2, wherein the amino acid sequence of the extracellular antigen recognition domain of the chimeric antigen receptor has at least 90% of the sequence of any one of SEQ ID NOS: 31-35. Homology. A pharmaceutical composition comprising the immune effector cell of any one of claims 1-20. Use of the immune effector cells according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 21 for the preparation of a medicament for preventing or treating a tumor or a pathogenic microorganism infection. A chimeric protein characterized in that the chimeric protein comprises an extracellular domain of a TGF-beta receptor and an intracellular signal domain of a receptor of an IL-2 family protein. The chimeric protein according to claim 23, wherein the chimeric protein comprises an intracellular signal of an extracellular domain of TGF-beta receptor II and an alpha subunit of a receptor of any one of the IL-2 family proteins area. The chimeric protein according to claim 23, wherein the chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular signal domain of IL-2RG. The chimeric protein according to any one of claims 23 to 25, wherein the extracellular domain of the TGF-beta receptor and the intracellular signal domain of the receptor of the IL-2 family protein are wild type. Or mutant. The chimeric protein according to claim 23, wherein The amino acid sequence of the extracellular domain of TGF-β receptor I has at least 90% homology with the amino acid sequence of SEQ ID NO: 4; The amino acid sequence of the extracellular domain of TGF-beta receptor II has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:12. The chimeric protein according to claim 25, wherein the amino acid sequence of the intracellular domain of IL-2RG has at least 90% homology with the amino acid sequence of SEQ ID NO: 8. The chimeric protein according to claim 24, wherein The amino acid sequence of the intracellular domain of IL-2Rβ has at least 90% homology with the amino acid sequence of SEQ ID NO: 16; The amino acid sequence of the intracellular domain of IL-7R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO:20; The amino acid sequence of the intracellular domain of IL-21R has at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 24. The chimeric protein according to claims 23-29, wherein the extracellular domain and the intracellular signal domain have a transmembrane domain; Preferably, the transmembrane domain is a transmembrane domain of a receptor for an IL-2 family protein. The chimeric protein according to any one of claims 23-29, wherein the amino acid sequence of the chimeric protein has at least 90% of the amino acid sequence shown in any one of SEQ ID NO: 27, 28, 29, or Homology Preferably, the amino acid sequence of the chimeric protein is as set forth in any one of SEQ ID NOs: 27, 28, 29, or 30. The chimeric protein according to claim 23, wherein the chimeric protein comprises a first chimeric protein and a second chimeric protein, The first chimeric protein comprises an extracellular domain of TGF-beta receptor I and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein ;with The second chimeric protein comprises an extracellular domain of TGF-beta receptor II and an intracellular domain of an intracellular signal domain selected from the intracellular signal domain of IL-2RG or the alpha subunit of a receptor of an IL-2 family protein .

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