CN109321530B - Safe chimeric antigen receptor T cell and application thereof - Google Patents

Abstract

The invention relates to a safe chimeric antigen receptor T cell and application thereof, and particularly provides a first CAR expressing a targeting HLA binding domain and a second CAR expressing a targeting tumor antigen. The engineered immune cells of the invention can selectively kill tumor cells and can also protect normal cells from killing.

Description

Safe chimeric antigen receptor T cell and application thereof

Technical Field

The invention relates to the field of immunotherapy, in particular to a safe chimeric antigen receptor T cell and application thereof.

Background

Tumor immunotherapy is considered as a fourth tumor treatment mode after surgery, radiotherapy and chemotherapy, and cellular immunotherapy is an extremely important component of immunotherapy. T/NK cells expressing Chimeric Antigen Receptors (CAR) in a transgenic manner, namely CAR-T/CAR-NK are the most widely applied cell types internationally, and the cells can specifically recognize targets on the surface of tumor cells, are activated and specifically kill target cells.

The existing CAR-T cell product is prepared by introducing CAR genes providing activation signals into T cells, only endows the T cells with simple recognition, activation and killing functions, and generates serious side effects. At present, the CAR-T recognizes target spots as tumor-associated antigens or specific biomarkers of a certain cell type, and these target spots are also expressed on normal cells, so that the CAR-T cells can target normal tissue cells to generate off-target effect (on-target off tumor), which causes fatal complications. Such as CAIX-CART, to treat metastatic renal cancer, resulting in targeting of normal bile duct cells, causing cholangitis; her2-CAR-T treats metastatic colorectal cancer, and as a result, targets lung epithelial cells, causing pulmonary edema, respiratory failure; CD19-CAR-T treats B cell hematological tumors and as a result targets normal B cells, causing B cell dysplasia.

Therefore, there is an urgent need in the art to develop a safe chimeric antigen receptor T cell that has a killing effect only on tumor cells, but does not kill normal cells.

Disclosure of Invention

The invention aims to provide a safe chimeric antigen receptor T cell which only has a killing effect on tumor cells and does not kill normal cells.

In a first aspect the invention provides an engineered immune cell comprising a first CAR expressing a targeting HLA binding domain and a second CAR targeting a tumour antigen,

and the first CAR has the structure shown in formula I:

L1-T1-Z1-Z2-TM1-C1 (I)

in the formula (I), the compound is shown in the specification,

l1 is an optional signal peptide sequence;

t1 is an HLA binding domain comprising an extracellular segment of a killer cell immunoglobulin-like receptor (KIR);

z1 is a null or hinge region;

z2 is a null or spacer domain (or spacer region);

TM1 is a transmembrane domain;

c1 is an intracellular T cell inhibitory signaling domain;

the structure of the second CAR is shown as formula II:

L2-T2-Z3-TM2-C2-CD3ζ (II)

in the formula (I), the compound is shown in the specification,

l2 is an optional signal peptide sequence;

t2 is a tumor antigen binding domain; and

z3 is a null or hinge region;

TM2 is a transmembrane domain;

c2 is a costimulatory signal molecule;

CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ;

and in each of the above formulae, each "-" is independently a linker peptide or a peptide bond.

In another preferred embodiment, the engineered immune cell further comprises a third CAR expressing a third antigen binding domain selected from the group consisting of: OPCML, HYAL2, DCC, SMAR1, E-cadherin, or a combination thereof.

In another preferred embodiment, the third CAR has the structure shown in formula III:

L3-T3-Z4-Z5-TM3-C3 (III)

in the formula (I), the compound is shown in the specification,

l3 is an optional signal peptide sequence;

t3 is a third antigen binding domain selected from the group consisting of: OPCML, HYAL2, DCC, SMAR1, E-cadherin, or a combination thereof;

z4 is a null or hinge region;

z5 is a null or spacer domain;

TM3 is a transmembrane domain;

c3 is an intracellular T cell inhibitory signaling domain;

and each "-" is independently a linker peptide or a peptide bond.

In another preferred embodiment, the first CAR, second CAR and optional third CAR are localised to the cell membrane of the immune cell.

In another preferred embodiment, the first CAR, the second CAR and optionally the third CAR are expressed on the cell membrane of the immune cell.

In another preferred embodiment, the first CAR, the second CAR and optionally the third CAR are recombinantly expressed.

In another preferred embodiment, the first CAR, the second CAR and optionally the third CAR are expressed from a vector.

In another preferred embodiment, the first CAR, the second CAR and optionally the third CAR are exogenous or endogenous.

In another preferred embodiment, the HLA binding domain further comprises a Fab, scFv, ligand, specific ligand and/or multivalent ligand.

In another preferred embodiment, the HLA binding domain comprises an HLA-C1 binding domain.

In another preferred embodiment, the HLA binding domain comprises the extracellular segment of killer immunoglobulin-like receptor (KIR) KIR2DL 2.

In another preferred embodiment, each of L1, L2 and L3 is independently a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred embodiment, each of Z1, Z3 and Z4 is independently a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

In another preferred embodiment, said Z2 and Z5 are spacer domains selected from the group consisting of: CD8, CD28, or a combination thereof.

In another preferred embodiment, the C1 and C3 are each independently selected from the group consisting of: intracellular fragments of programmed death factor-1 (PD-1), CTLA-4, LAG-3, 2B4, BTLA, TIM-3, or combinations thereof.

In another preferred embodiment, TM1, TM2, and TM3 are each independently a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CTLA-4, PD-1, LAG-3, 2B4, BTLA, or a combination thereof.

In another preferred embodiment, C2 is a costimulatory signaling molecule for a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

In another preferred embodiment, the tumor antigen binding domain is an antibody or antigen binding fragment.

In another preferred embodiment, the antigen binding fragment is a Fab or scFv or a single domain antibody sdFv.

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

(i) a chimeric antigen receptor T cell (CAR-T cell);

(ii) chimeric antigen receptor NK cells (CAR-NK cells); or

(iii) Exogenous T Cell Receptor (TCR) T cells (TCR-T cells).

In another preferred embodiment, the immune cells are autologous.

In another preferred embodiment, the immune cells are non-autologous.

In another preferred embodiment, the amino acid sequence of the first CAR is as shown in SEQ ID No. 5.

Furthermore, the invention provides the amino acid sequence of the first CAR and/or the second CAR of the invention, a nucleic acid sequence encoding the first CAR and/or the second CAR and a vector comprising the nucleic acid sequence.

In another preferred embodiment, a nucleic acid sequence encoding a first CAR of the invention is as shown in SEQ ID No. 6.

In a second aspect the invention provides a method of making an engineered immune cell comprising a first CAR expressing a targeting HLA binding domain and a second CAR targeting a tumour antigen, wherein the method comprises the steps of:

introducing into the immune cell a nucleic acid sequence encoding a first CAR that expresses a targeted HLA binding domain and a coding sequence encoding a second CAR that targets a tumor antigen, thereby obtaining an engineered immune cell, wherein the structures of the first CAR and the second CAR are as defined in the first aspect of the invention.

In another preferred embodiment, the introducing includes introducing simultaneously, sequentially or sequentially.

In another preferred embodiment, the method further comprises introducing into the immune cell a coding sequence encoding a third CAR that encodes a third antigen binding domain.

In a third aspect, the invention provides a pharmaceutical composition comprising an engineered immune cell according to the first aspect of the invention; and a pharmaceutically acceptable carrier, diluent or excipient.

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

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the engineered immune cell is (i) a chimeric antigen receptor T cell (CAR-T cell); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell).

In another preferred embodiment, the CAR-T cells or CAR-NK cells are present in the pharmaceutical composition at a concentration of 1X 10³-1×10⁶One cell/Kg body weight, preferably 1X 10⁴-1×10⁵One cell/Kg body weight.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs that selectively kill tumor cells (such as emerging antibody drugs, other CAR-T drugs, or chemotherapeutic drugs).

In a fourth aspect, the invention provides a use of the engineered immune cell of the first aspect of the invention for preparing a medicament or a preparation for selectively killing tumor cells.

In another preferred embodiment, the drug or formulation does not kill or substantially does not kill normal cells.

In another preferred embodiment, the engineered immune cell satisfies the formula: K1/K0 is more than or equal to 20, preferably 50, more preferably 100, more preferably 500, wherein K1 is the killing rate of the engineered immune cell to the tumor cell, and K0 is the killing rate of the engineered immune cell to the normal cell (preferably, the type of the normal cell is the same as that of the tumor cell, such as the hepatocyte vs hepatoma cell).

In another preferred embodiment, the normal cells include various normal cells other than tumor cells.

In another preferred embodiment, the tumor cell is derived from a tumor selected from the group consisting of: a B cell tumor, a T cell tumor, multiple myeloma, liver cancer, lung cancer, pancreatic cancer, skin cancer, colorectal cancer, head and neck tumor, nasopharyngeal cancer, esophageal cancer, breast cancer, cervical cancer, renal cancer, osteosarcoma, prostate cancer, or a combination thereof.

In a fifth aspect, the invention provides a kit for selectively killing tumor cells, the kit comprising a container, and an engineered immune cell according to the first aspect of the invention disposed within the container.

In another preferred embodiment, the kit further comprises a label or instructions for use.

In a sixth aspect, the present invention provides a method for selectively killing tumor cells, comprising:

administering to a subject in need thereof a safe and effective amount of an engineered immune cell according to the first aspect of the invention, or a pharmaceutical composition according to the third aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a schematic structural diagram of an iKP CAR;

FIG. 2 is a schematic diagram of the structure of an iKPt CAR;

FIG. 3 is a schematic structural view of iKP-19 CAR;

FIG. 4 is a schematic diagram of the structure of an iKPt-19 CAR;

FIG. 5 is CAR-T-19, CAR-T-iKP-19, CAR-T-iKPt-19 cell CAR positive rate;

FIG. 6A shows that CAR-T-iKP-19 kills CD19 +/HLA-tumor cells as well as CAR-T-19 and CAR-T-iKPt-19, and FIG. 6B shows that CAR-T-iKP-19 kills CD19 +/HLA-tumor cells or normal cells significantly less than CAR-T-19 and CAR-T-iKPt-19. The combination of FIGS. 6A and 6B shows that CAR-T-iKP-19 can selectively kill CD19 +/HLA-tumor cells and protect HLA + normal cells.

Detailed Description

The present inventors have extensively and deeply studied and, for the first time, unexpectedly found an engineered immune cell that targets both tumor antigens and HLA (including extracellular domain of killer immunoglobulin-like receptor (KIR)) and that selectively kills tumor cells and also protects normal cells from killing. On this basis, the present inventors have completed the present invention.

The present invention is representatively illustrated in detail for the engineered immune cells of the present invention, taking CAR-T cells as an example. The engineered immune cells of the invention are not limited to the CAR-T cells described above and below, and the engineered immune cells of the invention have the same or similar technical features and benefits as the CAR-T cells described above and below. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, the NK cell is identical to a T cell (or a T cell can replace an NK cell); when the immune cell is a T cell, the TCR is identical to the CAR (or the CAR can be replaced with a TCR).

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

By "endogenous" is meant that the nucleic acid molecule or polypeptide is normally expressed in a cell or tissue.

By "exogenous" is meant that the nucleic acid molecule or polypeptide is not endogenously present in the cell or is not present at a level sufficient to achieve the functional effect obtained upon overexpression. Thus, the term "exogenous" includes any recombinant nucleic acid molecule or polypeptide that is expressed in a cell, e.g., exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides.

Antibodies

The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

In the present invention, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. These fragments are also well known in the art and are often used in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragment F (ab')₂And Fab. F (ab')₂And Fab fragments lack the Fc fragment of intact antibody, clear more rapidly from circulation, and may have somewhat weaker nonspecific tissue binding of intact antibody (Wahl et al, J.Nucl. Med.24:316-325 (1983)). Antibodies of the invention include intact natural antibodies, bispecific antibodies; a chimeric antibody; fab; fab', single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies.

Preparation of antibodies

Any method suitable for producing monoclonal antibodies can be used to produce antibodies against the tumor antigens (e.g., CD19) and the HLA binding domains of the present invention, including extracellular domains of killer cell immunoglobulin-like receptors (KIRs). For example, an animal can be immunized with a homodimer or fragment thereof of a linked or naturally occurring tumor antigen (e.g., CD19) and the HLA binding domain, including extracellular segments of killer cell immunoglobulin-like receptors (KIRs). Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of tumor antigen (e.g., CD19) and the HLA binding domain (including extracellular segments of killer cell immunoglobulin-like receptor (KIR)) may be used as an immunogen (antigen) for generating non-human antibodies specific for tumor antigens (e.g., CD19) and the HLA binding domain (including extracellular segments of killer cell immunoglobulin-like receptor (KIR)), which antibodies are screened for biological activity. The challenge immunogen may be a full-length mature human tumor antigen (e.g., CD19) and the HLA binding domain (including extracellular segments of killer cell immunoglobulin-like receptors (KIRs)), include natural homodimers, or a peptide containing a single/multiple epitope. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculovirus vectors, plasmids, and non-viral vectors.

Fully humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. Optimization of the sequence of the essential constant domains to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.

Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell is any of various host cells conventionally used in the art, provided that the above recombinant expression vector is stably self-replicating and the nucleic acid carried thereby can be efficiently expressed. In particular, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.

Preferred host cells include e.coli TG1 or BL21 cells (expressing single chain antibodies or Fab antibodies), or CHO-K1 cells (expressing full length IgG antibodies).

The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

CD19

CD19 is expressed on B lineage cells (excluding mature plasma cells) and follicular dendritic cells, CD19 is an important signaling molecule that regulates the growth activation and activation of B lymphocytes, plays an important role in regulating the signaling threshold of B lymphocyte antigen receptors or other surface receptors, is an important membrane antigen associated with B lymphocyte differentiation, activation, proliferation and antibody production, and is the best marker for diagnosing B lymphocyte lineage tumors and identifying B lymphocytes

HLA

HLA molecules, i.e., human leukocyte antigens, are widely expressed on the cell surface of various tissue types of human beings and can be divided into 3 types of I, II and III molecules, wherein the I and II molecules participate in the immune response process. HLA class I molecules are involved in presentation of exogenous antigens and tumor antigens, and can be classified into HLA-A, HLA-B, HLA-C3 subtypes, and HLA-C is expressed in more than 95% of Han population

In a preferred embodiment of the invention, the selected target is HLA-C.

Extracellular domain of killer cell immunoglobulin-like receptor (KIR)

KIR, a killer immunoglobulin receptor, is mainly expressed on the surface of NK cells and a few CD8+ T cells, is located on chromosome 19q13.4, and is classified into inhibitory and activating action types. It is divided into two and three (KIR2D and KIR3D) immunoglobulin-like extracellular regions according to structure, and can specifically recognize MHC-I molecules.

KIR molecules with long intracellular segments contain 2 immunotyrosine inhibitory motifs in their cytoplasmic domain, primarily transmitting inhibitory signals; the cytoplasmic domain of KIR molecules with short intracellular segments lacks this motif, but has a characteristically charged residue in the transmembrane region through which activating signals can be transmitted by coupling to other molecules containing immunotyrosine activating motifs.

In a preferred embodiment of the invention, the extracellular domain of KIR2DL2 molecule that binds HLA-C is selected.

Chimeric Antigen Receptor (CAR)

Chimeric immune antigen receptors (CARs) consist of an extracellular antigen recognition region, usually a scFv (single-chain variable fragment), a transmembrane region, and an intracellular costimulatory signal region. The design of CARs goes through the following process: the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single activation domain in the cell, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and the development is three-generation and four-generation CARs.

The extracellular domain of CARs recognizes a specific antigen and subsequently transduces this signal through the intracellular domain, causing activated proliferation, cytolytic toxicity and cytokine secretion of the cell, thereby clearing the target cell. Autologous cells from the patient (or a heterologous donor) are first isolated, activated and genetically engineered to produce immune cells for CAR production, and then injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by immune cells in a non-MHC restricted manner.

CAR-immune cell therapy has achieved very high clinical response rates in the treatment of hematological malignancies, which rates were previously unattainable by any therapeutic approach, and have triggered a hot surge of clinical research in the world.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and/or a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or a cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecules and/or the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the 4-1BB signaling domain and/or the CD3 zeta signaling domain combination.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refer to an Fab fragment, Fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest 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 a scFv (single-chain variable fragment). The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the invention, the scFv comprises an antibody, preferably a single chain antibody, that specifically recognizes the tumor highly expressed antigens CD47 and MSLN.

In the present invention, the scFv of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or similar properties as compared with the amino acid sequence of the scFv of the present invention to form a polypeptide.

In the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

In the present invention, the first and second CARs and optionally the third CAR are as described in the first aspect of the invention.

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 according to the first aspect of the invention, which can target both HLA binding domains (including extracellular segments of killer immunoglobulin-like receptors (KIRs)) and tumor antigens (such as CD 19).

The first CAR and the second CAR and optionally the third CAR according to the invention, when expressed, pass through and are localised on the cell membrane.

CAR-T cells have the following advantages over other T cell-based therapies: (1) the action process of the CAR-T cell is not limited by MHC; (2) given that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once it is completed; (3) the CAR can utilize tumor protein antigens and glycolipid non-protein antigens, so that the target range of the tumor antigens is expanded; (4) the use of patient autologous cells reduces the risk of rejection; (5) the CAR-T cell has an immunological memory function and can survive in vivo for a long time.

In the present invention, the first CAR of the invention is a safe chimeric antigen receptor inhibiting T cell activation, inhibitory KIR-PD-1 polymeric antigen receptor, abbreviated iKP CAR, comprising (i) a Human Leukocyte Antigen (HLA) binding domain comprising an extracellular segment of a killer immunoglobulin-like receptor (KIR); (ii) spacer domain (iii) transmembrane domain; and (iv) an intracellular T cell inhibitory signaling domain comprising an intracellular segment of programmed death factor-1 (PD-1).

When the T cells of the invention expressing the first CAR (i.e., the iKP CAR) recognize HLA, intracellular PD-1 signaling is activated, inhibiting the signaling of the second CAR (i.e., the CD19CAR), and the T cells are restored to a resting state. A novel CAR-T cell, CAR-T-iKP-19 for short, was prepared by introducing a lentiviral vector into a conventional T cell expressing a second CAR of the invention (e.g., a CD19-CAR-T cell).

In a preferred embodiment, when CAR-T-iKP-19 recognizes a B cell tumor, the signal of the second CAR of the invention (i.e., CD19CAR) is activated and the T cell is activated to exert a killing function due to the tumor cell expressing high CD19 protein and low or no HLA protein; when CAR-T-iKP-19 recognizes normal B cells, which express both high expression CD19 protein and HLA protein, the iKPCAR signal is activated, and by recruiting the phosphatase SHP-2, dephosphorylating the second CAR (e.g., CD19CAR) signal of the invention, the T cells return to resting state and are unable to kill normal B cells.

A third CAR of the invention can be used in combination with a first CAR of the invention to inhibit the signal of the second CAR (i.e. a CD19CAR) to restore the T cell to a quiescent state.

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 a CAR-NK cell according to the first aspect of the invention. The CAR-NK cells of the invention can target both HLA binding domains, including extracellular segments of killer immunoglobulin-like receptors (KIRs), and tumor antigens, such as CD 19.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. By engineering (genetically modifying) NK cells it is possible to obtain new functions, including the ability to specifically recognize tumor antigens and having an enhanced anti-tumor cytotoxic effect.

CAR-NK cells also have the following advantages compared to autologous CAR-T cells, for example: (1) directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) they release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) is easy to be amplified in vitro and can be developed into ready-made products. Otherwise, similar to CAR-T cell therapy.

Exogenous T cell antigen receptor

As used herein, a foreign T cell antigen receptor (TCR) is a TCR that is exogenously transferred into a T cell by means of genetic engineering, using lentivirus or retrovirus as a vector, by cloning the α chain and β chain of the TCR from a tumor-reactive T cell by gene transfer technique.

The exogenous TCR modified T cell can specifically recognize and kill tumor cells, and affinity of the T cell and tumor can be improved and anti-tumor effect can be improved by optimizing affinity of TCR and tumor specific antigen.

Preparation

The invention provides an engineered immune cell according to the first aspect of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁸One cell/Kg body weight, more preferably 1X 10⁴-1×10⁷One cell/Kg body weight.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and 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 Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target HLA binding domains (including extracellular segments of killer immunoglobulin-like receptors (KIRs)) and tumor antigens (such as CD19), when the engineered T cells of the invention recognize B cell tumors, the T cells are activated to exert a killing function due to the tumor cells highly expressing CD19 protein, under-expressing or not expressing HLA protein, CD19CAR signaling is activated; when the modified T cell recognizes the normal B cell, the normal B cell expresses high expression CD19 protein and HLA protein at the same time, the iKPCAR signal is activated, and the T cell is restored to a resting state by recruiting phosphatase SHP-2 and dephosphorylating the CD19CAR signal, so that the normal B cell can not be killed.

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

In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells directed against HLA binding domains, including extracellular segments of killer cell immunoglobulin-like receptors (KIRs), and tumor antigens (e.g., CD19) elicit specific immune responses against cells expressing HLA binding domains, including extracellular segments of killer cell immunoglobulin-like receptors (KIRs), and tumor antigens (e.g., CD 19).

Although the data disclosed herein specifically disclose lentiviral vectors comprising an scFv for an anti-HLA binding domain, including extracellular segments of a killer cell immunoglobulin-like receptor (KIR), an scFv for an anti-tumor antigen such as CD19, a hinge and transmembrane region, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and 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 compositions of the present invention are preferably formulated for intravenous administration.

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

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the 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 T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment 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 efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with 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, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.

The dosage 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 proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁶1 to 10¹⁰A subject modified T cell (e.g., CAR-T20 cell) is administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include:

(1) the engineered immune cells of the invention can simultaneously target HLA binding domains (including extracellular domains of killer immunoglobulin-like receptors (KIRs)) and tumor antigens (such as CD19), thereby selectively killing tumor cells and inhibiting the killing of normal cells.

(2) The invention endows the T cells with the self-regulation function by simultaneously introducing the activation signal and the inhibition signal into the T cells, namely, when the CAR-T cells identify tumor cells, the CAR-T cells play a killing function; when CAR-T cells recognize normal cells, the killing function is inhibited.

(3) The invention designs a safe chimeric antigen receptor inhibitor KIR-PD-1chimeric antigen receptor for T cell activation for the first time, which is referred to as iKP CAR for short, when the iKP CAR recognizes HLA, an intracellular PD-1 signal is activated, a signal of normal CAR is inhibited, and T cells are restored to a resting state. When the CAR-T-iKP-19 recognizes B cell tumor, the CD19 protein is highly expressed by tumor cells, the HLA protein is lowly expressed or not expressed by the tumor cells, the CD19CAR signal is activated, the T cells are activated, and the killing function is exerted; when CAR-T-iKP-19 recognizes normal B cells, which express both high expression CD19 protein and HLA protein, the iKPCAR signal is activated, restoring the T cells to resting state by recruiting the phosphatase SHP-2, dephosphorylating the CD19CAR signal, and failing to kill normal B cells.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

The invention mainly constructs 3 kinds of slow virus packaging plasmids which are pCDH-19, pCDH-iKP-19 and pCDH-iKPt-19(PD-1 deletion), and the pCDH-19 and pCDH-iKPt-19 are negative control plasmids of pCDH-iKP-19.

Example 1 plasmid construction

1.1 construction of 1.1iKP/iKPt CAR

1.1.1KIR extracellular segment sequence search

The amino acid sequence of KIR2DL2 extracellular recognition domain was found from the uniprot database (http:// www.uniprot.org /) (excluding the signal peptide):

HEGVHRKPSLLAHPGRLVKSEETVILQCWSDVRFEHFLLHREGKFKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHECRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVIGNPSNSWPSPTEPSSKTGNPRHLH(SEQ ID NO.:1)

the corresponding nucleotide sequence is:

CATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCGCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTGAGCACTTCCTTCTGCACAGAGAAGGGAAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAAGCCAACTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAATGTAGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGTTTCTGTCATAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCTAAAACCGGTAACCCCCGACACCTGCAC(SEQ ID NO.:2)

1.1.2PD-1 intracellular Signaling Domain sequence search

The amino acid sequence of the PD-1 intracellular signaling domain was found from the uniprot database (http:// www.uniprot.org /):

CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO.:3)

the corresponding nucleotide sequence is:

TGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTC(SEQ ID NO.:4)

1.1.3iKP/iKPt CAR construction

The KIR extracellular recognition structural domain and the PD-1 intracellular signaling structural domain are connected by using CD8hinge and CD8 transmembrane region sequences, and then a CD8 signal peptide is added at the N end to construct a complete chimeric antigen receptor iKP CAR, wherein the structure is shown in figure 1. Meanwhile, the PD-1 intracellular segment is truncated to construct a negative control chimeric antigen receptor iKPtCAR (PD-1 truncated), and the structure is shown in FIG. 2.

iKP CAR amino acid sequence:

MALPVTALLLPLALLLHAARPHEGVHRKPSLLAHPGRLVKSEETVILQCWSDVRFEHFLLHREGKFKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHECRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVIGNPSNSWPSPTEPSSKTGNPRHLHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO.:5)

the corresponding nucleotide sequence is:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCGCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTGAGCACTTCCTTCTGCACAGAGAAGGGAAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAAGCCAACTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAATGTAGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGTTTCTGTCATAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCTAAAACCGGTAACCCCCGACACCTGCACACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA(SEQ ID NO.:6)

iKPt CAR amino acid sequence:

MALPVTALLLPLALLLHAARPHEGVHRKPSLLAHPGRLVKSEETVILQCWSDVRFEHFLLHREGKFKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHECRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVIGNPSNSWPSPTEPSSKTGNPRHLHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO.:7)

the corresponding nucleotide sequence is:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCGCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTGAGCACTTCCTTCTGCACAGAGAAGGGAAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAAGCCAACTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAATGTAGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGTTTCTGTCATAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAACCAAGCTCTAAAACCGGTAACCCCCGACACCTGCACACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTGA(SEQ ID NO.:8)

1.2 construction of expression vectors pCDH-iKP-19 and pCDH-iKPt-19

The constructed iKPCAR/iKPtCAR fragment is connected to a vector pCDH-CD19 which expresses CD19CAR and is preserved in a laboratory through T2A, and the vector is named as pCDH-iKP-19 and pCDH-iKPt-19, and the structures of the vector are shown in figures 3 and 4.

Example 2 Lentiviral packaging

Lentiviruses packaged by three plasmids, namely pCDH-19, pCDH-iKP-19 and pCDH-iKPt-19, are named lenti-19, lenti-iKP-19 and lenti-iKPt-19.

2.1. Plasmid transfection

1) Placing the plasmid, PEI and Opti-MEM culture medium at room temperature for 5 min;

2) putting 436 μ l of Opti-MEM into a 1.5ml EP tube, adding 64 μ g of PEI, mixing uniformly, and standing at room temperature for 5 min;

3) taking 12 μ g of vector plasmid pCDH-19, pCDH-iKP-19, pCDH-iKPt-19, 8 μ g of psPA x 2, 4 μ g of pMD2.G, adding Opti-MEM to 500 μ l, standing at room temperature for 5 min;

4) adding the prepared PEI-Opti-MEM solution into the Opti-MEM containing the plasmid, and standing for 20min at room temperature;

5) slowly dropping 1ml of DNA/PEI mixture into a 293T culture dish paved the day before, gently mixing, incubating in an incubator at 37 ℃, replacing fresh culture medium after 6-8h, and putting into the incubator at 37 ℃ for further incubation.

2.2 Virus Collection and concentration

1) After plasmid transfection for 48h, collecting supernatant, adding 10ml of fresh culture medium, continuously culturing for 72h, collecting supernatant again, mixing with the supernatant collected for 48h, and placing in a refrigerator at 4 ℃ for later use;

2) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris;

3) the resulting supernatant was filtered through a 0.45 μm filter;

4) transferring the filtered virus supernatant into an ultracentrifuge tube, centrifuging for 2h at 25000 r, diluting with PBS (1/100) in the volume of the supernatant, repeatedly blowing and transferring into a sealed centrifuge tube for overnight standing at 4 ℃;

5) the virus solution was dispensed to appropriate volumes, stored at-80 ℃ and 200. mu.l virus was titered.

2.3 Virus titre assay

1) Digesting 293T cells, centrifuging, counting, preparing cell suspension with serum-containing medium, and adjusting cell density to 4 × 10⁵Polybrene was added to 12. mu.g/ml per ml, and 0.5ml of cell suspension was added to each well of a 24-well plate;

2) viral supernatants were diluted with whole medium in the following proportions: 1: 3; 1: 9; 1: 27;

3) respectively adding 100 mul of virus stock solution and virus solution diluted according to different proportions into a 24-well plate inoculated with cells;

4) after 16h, the infection supernatant was discarded, and 0.5ml of fresh whole medium was added;

5) after 48 hours, detecting the target gene expression of the infected cells in a flow mode;

6) calculate titer, titer 2 x 10⁵Infection efficiency fold dilution.

The results are as follows:

after the viruses are collected and concentrated, the titer detection shows that the titer of lentiviruses of lenti-19, lenti-iKP-19 and lenti-iKPt-19 is 3 x 10 respectively⁸、1*10⁸、1.2*10⁸。

Example 3CAR-T cell preparation

Lentivirus lenti-19, lenti-iKP-19 and lenti-iKPt-19 are used for infecting human primary T cells to respectively obtain three CAR-T cells which are respectively named as CAR-T-19, CAR-T-iKP-19 and CAR-T-iKPt-19.

d0, collecting fresh cord blood, centrifuging by density gradient to separate PBMC, performing immunomagnetic bead screening with CD4/CD8 magnetic beads, and collecting positive cells 1 × 10⁶Inoculation is carried out at a density of/ml, X-vivo15 medium containing 10% FBS and 100U/ml IL-2 is added, and then TransAct is added according to a ratio of 1: 100;

d2 collecting T cells, centrifuging to change the culture medium, adding three kinds of lentiviruses including pCDH-19, pCDH-iKP-19 and pCDH-iKPt-19 into the culture medium according to the MOI ratio of 10:1, centrifuging to change the culture medium after 16h, adding fresh culture medium, and continuing to culture.

d6 cells were collected for flow detection.

The results are shown in FIG. 5. The results show that CAR-T-19, CAR-T-iKP-19, CAR-T-iKPt-19 were successfully prepared.

Example 4 target cell selection

The invention mainly screens 3 kinds of target cells, Daudi is B lymphocyte tumor cell line, CD19 positive/HLA negative; raji is B lymphocyte tumor cell line, positive CD 19/positive HLA; b cells were normal B lymphocytes, positive for CD 19/HLA positive.

Example 5 Selective killing of target cells by CAR-T-iKP-19

5.1 target cell labeling

Preparation of Single cell suspensions, 1 x 10⁶And/ml. 2ml of PBS was added and washed twice by centrifugation, and the serum was washed off.

Resuspend cells with PBS, adjust cell density to 2 x 10⁶And/ml. Adding equal volume of 10 mu M eFluor670 reagent, vortexing the cells, and incubating for 10 minutes at 37 ℃ in the dark; adding 4-5 times volume of pre-cooled complete culture medium of 10% serum,incubate 5 minutes on ice; complete medium was washed 3 times. Daudi, Raj i, B are labeled as eFluor 670.

5.2 Mixed culture of target cells and Effector cells

The Daudi, Raji and B stained with the eFluor670 were each 1 × 10⁵Number of wells inoculated into 48-well plates;

three CARTs of CAR-T-19, CAR-T-iKP-19 and CAR-T-iKPt-19 and T cells not infected by virus are mixed according to the effective target ratio of 1:5, 1:1, 5: 1, inoculating the three target cells, wherein each group is provided with three repeats, and each hole is filled with 1ml of solution;

culturing the mixed cell culture plate in an incubator at 37 ℃ for 4 h;

after 4h, all cells in each well were collected, transferred to a flow tube, incubated with PI, and tested on the machine.

5.3 killing efficiency analysis

Selecting an FL4 channel on a flow cytometer to detect the eFluor670, and trapping all cells positive to the eFluor 670; after the eFluor670 positive cycle, selecting FL2 channel to perform PI staining detection, wherein the PI staining positive cell is the apoptosis target cell. According to the flow results, the killing efficiency of each group of CAR-T-19, CAR-T-iKP-19 and CAR-T-iKPt-19 on Daudi, Raji and B is calculated, as shown in FIGS. 6A and 6B. The results in FIG. 6A show that CAR-T-iKP-19 kills CD19+/HLA-Daudi with no killing selectivity consistent with CAR-T-iKPt-19; the results in FIG. 6B show that CAR-T-iKP-19 had a significantly reduced killing effect on CD19+/HLA + Raji compared to CAR-T-19, CAR-T-iKPt-19, showing a selective killing effect.

Discussion of the related Art

CAR-T cell therapy is currently the most promising therapy to combat cancer, and has led to significant advances in hematological tumor therapy over traditional radiotherapy, chemotherapy, and even bone marrow transplantation. However, CAR-T cell therapy, as an emerging cancer treatment technology, has many issues to be solved clinically. Wherein the off-target effect is one of the most worried side effects for doctors and patients (as mentioned above). The invention introduces a negative signal for inhibiting T cell activation into a clinically universal second-generation CAR structure to regulate the activity of CAR-T cells, namely a CAR for transmitting a T cell activation inhibitory signal is constructed and is co-expressed on the same T cell with a CAR for providing an activation signal. The negative signal CAR extracellular recognition domain is an extracellular segment of KIR2DL2 molecules (16 members in the KIR family), and the recognized target points are HLA molecules which are highly expressed in normal tissue cells and are lowly expressed in tumor cells. Accordingly, similar targets also include OPCML/HYAL2/DCC/SMAR1/E-cadherin, etc. (molecules that are highly expressed on the cell surface of normal tissue and less expressed on the cell surface of tumor cells). The intracellular signaling domain for transmitting negative signals is the intracellular segment of PD-1 molecule, and correspondingly, the negative signal molecules capable of similarly inhibiting T cell activation include TIM-3/LAG-3/CTLA-4 and other (containing immune receptor tyrosine inhibitory motif ITIM or immune receptor tyrosine switch motif ITSM) molecules. The B lymphocyte leukemia model and the CD19 target point selected in the embodiment of the invention can be expanded to other types of tumors and corresponding target points. The invention provides an innovative CAR-T cell which can recognize targets on normal tissue cells and tumor cells to perform signal self-regulation, can selectively kill the tumor cells and protect the normal tissue cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> university of east China

Shanghai Bangyao Biological Technology Co.,Ltd.

<120> safe chimeric antigen receptor T cell and application thereof

<130> P2018-0186

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 224

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 1

His Glu Gly Val His Arg Lys Pro Ser Leu Leu Ala His Pro Gly Arg

1 5 10 15

Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val

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Arg Phe Glu His Phe Leu Leu His Arg Glu Gly Lys Phe Lys Asp Thr

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Leu His Leu Ile Gly Glu His His Asp Gly Val Ser Lys Ala Asn Phe

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Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly Thr Tyr Arg Cys Tyr

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Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro

85 90 95

Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala

100 105 110

Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser Val Thr Leu Ser Cys

115 120 125

Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu Ser Arg Glu Gly Glu

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Ala His Glu Cys Arg Phe Ser Ala Gly Pro Lys Val Asn Gly Thr Phe

145 150 155 160

Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr Arg

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Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu Trp Ser Asn Ser Ser

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Asp Pro Leu Leu Val Ser Val Ile Gly Asn Pro Ser Asn Ser Trp Pro

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Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His

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<211> 672

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

catgagggag tccacagaaa accttccctc ctggcccacc caggtcgcct ggtgaaatca 60

gaagagacag tcatcctgca atgttggtca gatgtcaggt ttgagcactt ccttctgcac 120

agagaaggga agtttaagga cactttgcac ctcattggag agcaccatga tggggtctcc 180

aaagccaact tctccatcgg tcccatgatg caagaccttg cagggaccta cagatgctac 240

ggttctgtta ctcactcccc ctatcagttg tcagctccca gtgaccctct ggacatcgtc 300

atcacaggtc tatatgagaa accttctctc tcagcccagc cgggccccac ggttctggca 360

ggagagagcg tgaccttgtc ctgcagctcc cggagctcct atgacatgta ccatctatcc 420

agggaggggg aggcccatga atgtaggttc tctgcagggc ccaaggtcaa cggaacattc 480

caggccgact ttcctctggg ccctgccacc cacggaggaa cctacagatg cttcggctct 540

ttccgtgact ctccatacga gtggtcaaac tcgagtgacc cactgcttgt ttctgtcata 600

ggaaaccctt caaatagttg gccttcaccc actgaaccaa gctctaaaac cggtaacccc 660

cgacacctgc ac 672

<210> 3

<211> 97

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 3

Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln

1 5 10 15

Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr

20 25 30

Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val

35 40 45

Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser

50 55 60

Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro

65 70 75 80

Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro

85 90 95

Leu

<210> 4

<211> 291

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

tgctcccggg ccgcacgagg gacaatagga gccaggcgca ccggccagcc cctgaaggag 60

gacccctcag ccgtgcctgt gttctctgtg gactatgggg agctggattt ccagtggcga 120

gagaagaccc cggagccccc cgtgccctgt gtccctgagc agacggagta tgccaccatt 180

gtctttccta gcggaatggg cacctcatcc cccgcccgca ggggctcagc tgacggccct 240

cggagtgccc agccactgag gcctgaggat ggacactgct cttggcccct c 291

<210> 5

<211> 411

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 5

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Arg Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Arg Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Lys Phe Lys Asp Thr Leu His Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Cys Arg Phe Ser Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Ile Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

245 250 255

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

260 265 270

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

275 280 285

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

290 295 300

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Cys Ser Arg Ala Ala Arg

305 310 315 320

Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro

325 330 335

Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln

340 345 350

Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu Gln

355 360 365

Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser Ser

370 375 380

Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro Leu

385 390 395 400

Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

405 410

<210> 6

<211> 1236

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcatgagg gagtccacag aaaaccttcc ctcctggccc acccaggtcg cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcagatgtca ggtttgagca cttccttctg 180

cacagagaag ggaagtttaa ggacactttg cacctcattg gagagcacca tgatggggtc 240

tccaaagcca acttctccat cggtcccatg atgcaagacc ttgcagggac ctacagatgc 300

tacggttctg ttactcactc cccctatcag ttgtcagctc ccagtgaccc tctggacatc 360

gtcatcacag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttctg 420

gcaggagaga gcgtgacctt gtcctgcagc tcccggagct cctatgacat gtaccatcta 480

tccagggagg gggaggccca tgaatgtagg ttctctgcag ggcccaaggt caacggaaca 540

ttccaggccg actttcctct gggccctgcc acccacggag gaacctacag atgcttcggc 600

tctttccgtg actctccata cgagtggtca aactcgagtg acccactgct tgtttctgtc 660

ataggaaacc cttcaaatag ttggccttca cccactgaac caagctctaa aaccggtaac 720

ccccgacacc tgcacaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 780

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 840

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 900

tgtggggtcc ttctcctgtc actggttatc accctttact gctgctcccg ggccgcacga 960

gggacaatag gagccaggcg caccggccag cccctgaagg aggacccctc agccgtgcct 1020

gtgttctctg tggactatgg ggagctggat ttccagtggc gagagaagac cccggagccc 1080

cccgtgccct gtgtccctga gcagacggag tatgccacca ttgtctttcc tagcggaatg 1140

ggcacctcat cccccgcccg caggggctca gctgacggcc ctcggagtgc ccagccactg 1200

aggcctgagg atggacactg ctcttggccc ctctga 1236

<210> 7

<211> 314

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 7

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Arg Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Arg Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Lys Phe Lys Asp Thr Leu His Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Cys Arg Phe Ser Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Ile Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

245 250 255

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

260 265 270

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

275 280 285

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

290 295 300

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

305 310

<210> 8

<211> 945

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcatgagg gagtccacag aaaaccttcc ctcctggccc acccaggtcg cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcagatgtca ggtttgagca cttccttctg 180

cacagagaag ggaagtttaa ggacactttg cacctcattg gagagcacca tgatggggtc 240

tccaaagcca acttctccat cggtcccatg atgcaagacc ttgcagggac ctacagatgc 300

tacggttctg ttactcactc cccctatcag ttgtcagctc ccagtgaccc tctggacatc 360

gtcatcacag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttctg 420

gcaggagaga gcgtgacctt gtcctgcagc tcccggagct cctatgacat gtaccatcta 480

tccagggagg gggaggccca tgaatgtagg ttctctgcag ggcccaaggt caacggaaca 540

ttccaggccg actttcctct gggccctgcc acccacggag gaacctacag atgcttcggc 600

tctttccgtg actctccata cgagtggtca aactcgagtg acccactgct tgtttctgtc 660

ataggaaacc cttcaaatag ttggccttca cccactgaac caagctctaa aaccggtaac 720

ccccgacacc tgcacaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 780

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 840

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 900

tgtggggtcc ttctcctgtc actggttatc accctttact gctga 945

Claims (18)

1. An engineered immune cell comprising a first CAR expressing an HLA binding domain and a second CAR targeting a tumor antigen,

and the first CAR has the structure shown in formula I:

L1-T1-Z1-Z2-TM1-C1 (I)

in the formula (I), the compound is shown in the specification,

l1 is an optional signal peptide sequence;

t1 is an HLA binding domain comprising an extracellular segment of killer cell immunoglobulin-like receptor (KIR), which is an extracellular segment of KIR2DL2 of killer cell immunoglobulin-like receptor (KIR);

z1 is a null or hinge region;

z2 is a null or spacer sequence region;

TM1 is a transmembrane domain;

c1 is an intracellular T cell inhibitory signaling domain, and C1 is selected from the group consisting of: an intracellular segment of programmed death factor-1 (PD-1), CTLA-4, LAG-3, 2B4, BTLA, TIM-3, or a combination thereof;

the structure of the second CAR is shown as formula II:

L2-T2-Z3-TM2-C2-CD3ζ (II)

in the formula (I), the compound is shown in the specification,

l2 is an optional signal peptide sequence;

t2 is a tumor antigen binding domain; and

z3 is a null or hinge region;

TM2 is a transmembrane domain;

c2 is a costimulatory signal molecule;

CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ and the second CAR is a CD 19-targeted CAR;

and in each of the above formulae, each "-" is independently a linker peptide or a peptide bond.

2. The engineered immune cell of claim 1, further comprising a third CAR that expresses a third antigen binding domain selected from the group consisting of: OPCML, HYAL2, DCC, SMAR1, E-cadherin, or a combination thereof.

3. The engineered immune cell of claim 2, wherein the third CAR has a structure according to formula III:

L3-T3-Z4-Z5-TM3-C3 (III)

in the formula (I), the compound is shown in the specification,

l3 is an optional signal peptide sequence;

t3 is a third antigen binding domain selected from the group consisting of: OPCML, HYAL2, DCC, SMAR1, E-cadherin, or a combination thereof;

z4 is a null or hinge region;

z5 is a null or spacer sequence region;

TM3 is a transmembrane domain;

c3 is an intracellular T cell inhibitory signaling domain;

and each "-" is independently a linker peptide or a peptide bond.

4. The engineered immune cell of claim 1, wherein the HLA binding domain comprises an HLA-C1 binding domain.

5. The engineered immune cell of claim 1 or 3, wherein each of L1, L2, and L3 is independently a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

6. The engineered immune cell of claim 1 or 3, wherein each of Z1, Z3, and Z4 is independently a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

7. The engineered immune cell of claim 1 or 3, wherein Z2 and Z5 are spacer domains selected from the group consisting of: CD8, CD28, or a combination thereof.

8. The engineered immune cell of claim 3, wherein said C3 is selected from the group consisting of: intracellular fragments of programmed death factor-1 (PD-1), CTLA-4, LAG-3, 2B4, BTLA, TIM-3, or combinations thereof.

9. The engineered immune cell of claim 1 or 3, wherein each of TM1, TM2, and TM3 is independently a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon (T cell surface glycoprotein), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CTLA-4, PD-1, LAG-3, 2B4, BTLA, or a combination thereof.

10. The engineered immune cell of claim 1, wherein C2 is a costimulatory signaling molecule for a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1, ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

11. The engineered immune cell of claim 10, wherein the LFA-1 comprises CD11a/CD 18.

12. The engineered immune cell of claim 1, wherein the tumor antigen binding domain is an antibody or antigen binding fragment.

13. The engineered immune cell of claim 1, wherein the amino acid sequence of the first CAR is as set forth in SEQ ID No. 5.

14. A method of making an engineered immune cell comprising a first CAR that expresses a targeted HLA binding domain and a second CAR that is targeted to a tumor antigen, wherein the method comprises the steps of:

introducing into the immune cell a nucleic acid sequence encoding a first CAR that expresses a targeted HLA binding domain and a coding sequence encoding a second CAR that targets a tumor antigen, thereby obtaining an engineered immune cell, wherein the structures of the first CAR and the second CAR are as defined in claim 1.

15. The method of claim 14, further comprising expressing a coding sequence that introduces a third CAR encoding a third antigen binding domain into the immune cell.

16. A pharmaceutical composition comprising the engineered immune cell of claim 1; and a pharmaceutically acceptable carrier, diluent or excipient.

17. Use of the engineered immune cell of claim 1 for the preparation of a medicament or formulation for selective killing of tumor cells.

18. A kit for selectively killing tumor cells, comprising a container, and the engineered immune cell of claim 1 disposed within the container.

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