CN110818802A

CN110818802A - Chimeric T cell receptor STAR and application thereof

Info

Publication number: CN110818802A
Application number: CN201810898720.2A
Authority: CN
Inventors: 刘玥; 王嘉盛; 刘光娜; 赵学强; 林欣
Original assignee: Sino-American iCELL (Shanghai) Biotechnology Co Ltd; Huaxia Yingtai (beijing) Biotechnology Co Ltd; Tsinghua University
Current assignee: Tsinghua University; Bristar Immunotech Ltd
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2020-02-21
Anticipated expiration: 2038-08-08
Also published as: CN110818802B; WO2020029774A1

Abstract

The invention discloses a chimeric T Cell Receptor (STAR), a related preparation, a medicine or application thereof in preparation of a Cell medicine, and further relates to the preparation or a medicine composition for treating corresponding diseases, such as tumors or infectious diseases.

Description

Chimeric T cell receptor STAR and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an antibody-T cell receptor chimeric receptor, a construction method and application thereof, including treatment and diagnosis of diseases.

Background

Chimeric antigen receptor T cell therapy (CAR-T) and T cell receptor therapy (TCR-T) are both types of leading-edge gene therapy that utilize the patient's own T lymphocytes to treat cancer. They are capable of expressing specific receptors, target-recognizing specific cells such as tumor cells, have received extensive attention and research, transitioning from the first basic immunological studies to clinical applications. Recent advances in synthetic biology, immunology and genetic engineering technologies have made it possible to synthesize engineered T cells with enhanced specific function.

CAR-T is an antigen that recognizes the surface of tumor cells using antibody fragments that are capable of binding to a specific antigen. In recent years, CD19 antigen-specific CAR-T cells have shown sustained disease remission in clinical trials for the treatment of B cell leukemia and lymphoma. Chimeric Antigen Receptors (CARs) confer the ability of T cells to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets relative to native T cell surface receptor TCRs. Currently, CAR-T technology has significant efficacy in the treatment of acute leukemias and non-hodgkin's lymphomas, and is considered to be one of the most promising modes of tumor treatment.

Unlike CAR-T, tcr (T cell receptor) is a molecule that specifically recognizes antigens and mediates immune responses on the surface of T cells. The TCR primarily recognizes antigenic molecule polypeptides presented by histocompatibility complex molecules. In recent years, the search for TCR therapy has been relatively less attractive due to the great clinical success of CAR-T cell therapy. However, TCR-T has a clear advantage over CAR-T in treating solid tumors because the target antigen for CAR-T cell therapy is a cell surface protein, whereas TCR-T recognizes MHC molecules that are capable of presenting peptide chains derived from cell surface and intracellular proteins, and thus are capable of targeting a wider variety of antigens.

It is known that most Tumor Associated Antigens (TAAs) are self-antigens, and due to the selection mechanism and tolerance mechanism of thymus, the affinity of most T Cell Receptors (TCRs) of T lymphocytes generated by the body against these antigens is low, thereby limiting their tumor recognition and killing effect. The cloned TCR (or chimeric receptor) with high affinity for identifying TAA is transferred to T lymphocyte by transgenic technology, so that the redirected T cell without tumor identification capability can effectively identify and kill tumor cells in vitro and in vivo. TCR is a characteristic marker of all T cell surfaces, and binds non-covalently to CD3 (epsilon, delta, gamma, zeta) to form the TCR-CD3 complex.

The TCR is a heterodimer composed of two different peptide chains, each peptide chain can be divided into a variable region (V region) and a constant region (C region), wherein the constant region comprises an extracellular region, a transmembrane region and an intracellular terminal, and is characterized in that the intracellular region is short, the TCR molecule belongs to an immunoglobulin superfamily, and the antigen specificity of the TCR molecule exists in the V region, the TCR is divided into two types, namely TCR1 and TCR2, TCR1 consists of two chains of gamma and delta, TCR2 consists of two chains of α and β, 90% -95% of T cells express TCR2 in peripheral blood, and any T cell only expresses one of TCR2 or TCR 1.

Since a naturally occurring TCR is a membrane protein and is stabilized by its transmembrane region, it is very difficult to obtain a highly stable TCR which retains the ability to specifically bind to its original ligand (i.e., pMHC) for expression of soluble TCRs in bacteria, as described in WO 99/18129. Some documents describe truncated forms of TCRs comprising only the extracellular region or only the extracellular and cytoplasmic regions, although such TCRs are recognized by TCR-specific antibodies, but the yields are low and at low concentrations they do not recognize the major histocompatibility complex-peptide complex, indicating that they are easily denatured and not stable enough.

The TCR-T technology has the advantages that: traditional adoptive immunotherapy only increases the number of effector cells, does not improve the specificity of effector cells, and has low affinity even if the effector cells can bind to tumor cells. The TCR-T technology directly modifies a probe (TCR) of a T cell, which is combined with a tumor antigen, strengthens the specific recognition process of the T cell for the tumor cell, improves the affinity of the T lymphocyte for the tumor cell, and leads the original T cell without tumor recognition capability to effectively recognize and kill the tumor cell in vitro and in vivo. In a word, the TCR-T cell therapy increases the number of T lymphocytes and improves the killing property of the T lymphocytes on tumor cells, thereby achieving good tumor treatment effect.

In the current TCR-T therapy, endogenous TCRs generally need to be isolated and engineered, introduced into new T cells and infused back into the human body, and the number of T cells with the ability to target cancer cells will be greatly increased, and thus, the recognition and attack of various solid tumors and hematological tumors is expected.

Therefore, how to modify TCR genes to enable a TCR α chain and a β chain to be correctly paired on the surface of a T cell, and the expression efficiency and the affinity of the TCR chains are enhanced, meanwhile, side effects are avoided, and the safety is improved to become one of hot spots of TCR gene therapy in recent years.

Disclosure of Invention

The present invention solves the above technical problems in the prior art, and provides a chimeric T Cell Receptor (STAR) that specifically binds to a target Antigen, the chimeric T Cell Receptor comprising:

a) a first peptide chain obtained by fusing an antibody heavy chain variable region with a T Cell Receptor (TCR) first subunit constant region; and the combination of (a) and (b),

b) a second peptide chain obtained by fusing the variable region of the antibody light chain with a constant region of a second subunit of a T cell receptor;

wherein the antibody heavy chain variable region and antibody light chain variable region specifically bind to an epitope of the target antigen.

In some embodiments, the chimeric T cell receptor according to the foregoing is (1) a β chain for the second subunit of the T cell receptor when the first subunit of the T cell receptor is an α chain, or (2) a α chain for the second subunit of the T cell receptor when the first subunit of the T cell receptor is a β chain, or (3) a δ chain for the second subunit of the T cell receptor when the first subunit of the T cell receptor is a γ chain, or (4) a γ chain for the second subunit of the T cell receptor when the first subunit of the T cell receptor is a δ chain.

Specifically, the first peptide chain and the second peptide chain are bound by a disulfide bond after being expressed in a T cell.

In some embodiments, the chimeric T cell receptor first subunit constant region and the T cell receptor second subunit constant region are of human or murine origin, including different protein subtypes.

In some embodiments, the chimeric T cell receptor can be modified with any amino acid sequence, including but not limited to amino acid point mutation modification, polypeptide fragment substitution modification, to reduce mismatches with endogenously expressed T cell receptors.A.i.e., TCR α chain, whose constant region has amino acid 48 mutated to cysteine and whose constant region has amino acid 57 mutated to cysteine, allows for a disulfide bond linkage between the first and second polypeptides;

specifically, (1) the first subunit of the T cell receptor is a TCR α chain, the 48 th amino acid of the constant region of the first subunit is mutated into cysteine, and/or the second subunit of the T cell receptor is a TCR β chain, the 57 th amino acid of the constant region of the second subunit is mutated into cysteine, or (2) the first subunit of the T cell receptor is a TCR α chain, the 85 th amino acid of the constant region of the first subunit is mutated into alanine, and/or the second subunit of the T cell receptor is a TCR β chain, and the 88 th amino acid of the constant region is mutated into glycine.

In some embodiments, the target antigen is a tumor-specific antigen or a virus-specific antigen. In particular the target antigen is selected from the group consisting of CD19, CD20, EGFR, Her2, PSCA, CD123, CEA (carcinoembryonic antigen), FAP, CD133, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, VEGFR, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, CD 171. Preferably, the antigen may be CD19, CD20, EGFR, Her 2.

Specifically, in some embodiments, the antibody, antibody heavy chain variable region, or antibody light chain variable region is derived from IMCC225 (Cetuximab, Cetuximab/Cetux), rituximab (rituximab), Ofatumumab (OFA, Ofatumumab), CD19 monoclonal antibody (FMC63), Avastin (bevacizumab), BEC2 (adoumumab), Bexxar (tositumomab), Campath (alemtuzumab), Herceptin (trastuzumab), lymphocid (epratuzumab), MDX-210, Mylotarg (gemumab ozomicin), mab 17-1A (ibritumomab), theragyn (pemtumumab), Zamyl, Zevalin (ibritumomab tiuj) or a high affinity antibody obtained by screening. Preferably, the antibody thereof comprises an antigen binding fragment selected from the group consisting of Fab, F (ab ') 2, Fab', scFv, Fv, VH, VL.

In some embodiments, the target antigen-associated disease is a cancer or a disease associated with a viral infection. In particular, the cancer is selected from the group consisting of: adrenocortical, bladder, breast, cervical, biliary, colorectal, esophageal, glioblastoma, glioma, hepatocellular, head and neck, renal, leukemia, lymphoma, lung, melanoma, mesothelioma, multiple myeloma, pancreatic, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian, prostate, sarcoma, gastric, uterine and thyroid cancers; or, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr virus (EBV), Hepatitis B Virus (HBV), Kaposi's sarcoma-associated herpes virus (KSHV), Human Papilloma Virus (HPV), Molluscum Contagiosum Virus (MCV), human T-cell leukemia virus 1(HTLV-1), HIV (human immunodeficiency virus), and Hepatitis C Virus (HCV).

The chimeric T cell receptor of the invention, the first and second polypeptides form a complex with the endogenous CD3 subunit (epsilon, delta, gamma, zeta) of the T cell.

In some embodiments, the antibody heavy chain variable region and the light chain variable region are derived from the VH and VL of IMCC225 (Cetuximab, Cetuximab/Cetux), rituximab (rituximab), Ofatumumab (Ofatumumab), CD19 monoclonal antibody FMC 63.

Preferably, the nucleotide sequence of the Cetux VH is SEQ ID NO. 3, the amino acid sequence of the Cetux VL is SEQ ID NO. 13, the nucleotide sequence of the Cetux VL is SEQ ID NO. 4, and the amino acid sequence of the Cetux VL is SEQ ID NO. 14.

Preferably, the nucleotide sequence of FMC63-VH is SEQ ID NO. 5, the amino acid sequence thereof is SEQ ID NO. 15, the nucleotide sequence of FMC63-VL is SEQ ID NO. 6, and the amino acid sequence thereof is SEQ ID NO. 16; .

Preferably, the nucleotide sequence of the OFA-VH is SEQ ID NO. 9, the amino acid sequence thereof is SEQ ID NO. 19, the nucleotide sequence of the OFA-VL is SEQ ID NO. 10, and the amino acid sequence thereof is SEQ ID NO. 20.

In some embodiments, the VH and VL derived from cetuximab, trastuximab, rituximab are fused to the TCR α chain or β chain, respectively, to obtain a VH-TCR α chain fusion or a VL-TCR β chain fusion.

Further preferably, the VH and VL of the monoclonal antibody FMC63 derived from IMCC225 (Cetuximab, Cetuximab/Cetux), rituximab (rituximab), Ofatumumab (Ofatumumab), CD19 are fused to the TCR α chain constant region or the β chain constant region, respectively, to obtain a VH-TCR α chain constant region fusion or a VL-TCR β chain constant region fusion.

Preferably, the two different fusions are joined by a furin-p2A stretch peptide sequence; preferably, the two different fusions are covalently bound by a disulfide bond after expression in a T cell. Further, the two different fusions form a complex with the endogenous CD3 subunit (epsilon, delta, gamma, zeta) of the T cell.

In addition, the invention provides a complex formed by the chimeric T cell receptor specifically binding to a target antigen, wherein the chimeric T cell receptor of any one of the chimeric T cell receptors forms a complex with a CD3 subunit (epsilon, delta, gamma and zeta) endogenously expressed by a T cell, and can mediate a T cell-related signal transduction pathway after being activated by the target antigen.

In some embodiments, the invention also provides a nucleic acid encoding the chimeric T cell receptor of any one of the preceding or the first and second polypeptides.

Specifically, the structure of the nucleic acid is as follows:

(1) sequentially comprises an antibody heavy chain variable region, a T Cell Receptor (TCR) α chain constant region extracellular section, a transmembrane region and an intracellular tail end, a linker, an antibody light chain variable region, a T Cell Receptor (TCR) β chain constant region extracellular section, a transmembrane region and an intracellular tail end, or,

(2) sequentially comprises an antibody heavy chain variable region, a T Cell Receptor (TCR) β chain constant region extracellular section, a transmembrane region and an intracellular tail end, a linker, an antibody light chain variable region, a T Cell Receptor (TCR) α chain constant region extracellular section, a transmembrane region and an intracellular tail end, or,

(3) sequentially comprises an antibody light chain variable region, a T Cell Receptor (TCR) α chain constant region extracellular section, a transmembrane region and an intracellular tail end, a linker, an antibody heavy chain variable region, a T Cell Receptor (TCR) β chain constant region extracellular section, a transmembrane region and an intracellular tail end, or,

(4) sequentially comprises an antibody light chain variable region, a T Cell Receptor (TCR) β chain constant region extracellular section, a transmembrane region and an intracellular tail end, a linker, an antibody heavy chain variable region, a T Cell Receptor (TCR) α chain constant region extracellular section, a transmembrane region and an intracellular tail end, or,

(5) replacing the T Cell Receptor (TCR) α chain in (1) - (4) with a T Cell Receptor (TCR) gamma chain and the T Cell Receptor (TCR) β chain with a nucleic acid corresponding to the T Cell Receptor (TCR) delta chain.

In some embodiments, the T Cell Receptor (TCR) α chain or β chain constant region is derived from a human TCR α chain or β chain constant region, and may also be derived from a murine TCR α chain or β chain constant region.

In some embodiments, the α and/or β chains of the TCR may be modified by amino acid point mutations, polypeptide fragment substitutions to reduce mismatches with endogenously expressed T cell receptors, preferably the α and/or β chains of the TCR are modified by cysteine point mutations, specifically, the 48 th amino acid of the constant region of the TCR α chain is mutated to cysteine and the 57 th amino acid of the constant region of the TCR β chain is mutated to cysteine, such that the first and second peptide chains are linked by adding a disulfide bond.

More preferably, the nucleotide sequence of the human TCR α chain constant region cysteine mutant is SEQ ID NO. 1, the amino acid sequence is SEQ ID NO. 11, the nucleotide sequence of the human TCR β chain constant region cysteine mutant is SEQ ID NO. 2, and the amino acid sequence is SEQ ID NO. 12.

In another embodiment, the amino acid sequence of the constant region of the human TCR γ chain is SEQ ID NO 21, the amino acid sequence of the constant region of the human TCR δ chain is SEQ ID NO 22, the amino acid sequence of the constant region of the murine TCR γ chain is SEQ ID NO 23 and the amino acid sequence of the constant region of the murine TCR δ chain is SEQ ID NO 24.

Furthermore, the natural human TCR α chain constant region first amino acid exists in four forms, the first amino acid is Asp, namely the polypeptide shown in SEQ ID NO:11, and the first amino acid is Asn, His or Tyr, so that the human TCR α chain constant region cysteine mutant can be selected from the polypeptides represented by the following sequences:

① has the amino acid sequence of SEQ ID NO. 31 and the corresponding nucleotide sequence of SEQ ID NO. 25;

② has the amino acid sequence of SEQ ID NO. 32 and the corresponding nucleotide sequence of SEQ ID NO. 26;

③ has the amino acid sequence of SEQ ID NO. 33 and the corresponding nucleotide sequence of SEQ ID NO. 27.

More preferably, the nucleotide sequence of the mouse TCR α chain constant region cysteine mutant is SEQ ID NO. 7, and the amino acid sequence is SEQ ID NO. 17, the nucleotide sequence of the mouse TCR β chain constant region cysteine mutant is SEQ ID NO. 8, and the amino acid sequence is SEQ ID NO. 18.

Furthermore, the natural mouse TCR α chain constant region first amino acid has four forms, wherein the first amino acid is Asn, namely the polypeptide shown in SEQ ID NO:17, and the first amino acid is Asp, His or Tyr, so the mouse TCR α chain constant region cysteine mutant can be selected from the polypeptides represented by the following sequences:

① has the amino acid sequence of SEQ ID NO. 34 and the corresponding nucleotide sequence of SEQ ID NO. 28;

② has an amino acid sequence of SEQ ID NO. 35 and a corresponding nucleotide sequence of SEQ ID NO. 29;

③ has the amino acid sequence as SEQ ID NO. 36 and the corresponding nucleotide sequence as SEQ ID NO. 30.

In some embodiments, the T Cell Receptor (TCR) α chain can be replaced with a T Cell Receptor (TCR) γ chain and the T Cell Receptor (TCR) β chain can be replaced with a T Cell Receptor (TCR) δ chain.

In some embodiments, the present invention also provides a vector comprising a nucleic acid encoding the chimeric T cell receptor of any one of the preceding or the first and second polypeptides. Preferably, the vector is a plasmid. More preferably, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

In some embodiments, according to any one of the chimeric T cell receptors described above, there is provided an effector cell expressing on its cell surface the chimeric T cell receptor of any of the foregoing or the foregoing complex. In some embodiments, the effector cell comprises a nucleic acid encoding the chimeric T cell receptor. In some embodiments, the T cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

In some embodiments, there is provided a pharmaceutical composition comprising a chimeric T cell receptor according to any one of the chimeric T cell receptors described above and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a nucleic acid encoding the chimeric T cell receptor according to any one of the above embodiments and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising effector cells expressing any one of the chimeric T cell receptors described above and a pharmaceutically acceptable carrier.

The chimeric T cell receptor of the invention and cells transfected with the chimeric T cell receptor of the invention can be provided in a pharmaceutical composition with a pharmaceutically acceptable carrier. The chimeric T cell receptors, complexes of chimeric T cell receptors, and cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.

The chimeric T cell receptors of the invention may be used alone or in combination or conjugation with a conjugate. The conjugates include a detectable label, a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MIR (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. Such vectors include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof. Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.

Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated may be an animal, in particular a human.

When the pharmaceutical composition of the present invention is used for practical treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, injections, oral agents and the like are exemplified. These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.

The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the polypeptides of the invention may be incorporated into pellets or microcapsules carried by a slow release polymer, which pellets or microcapsules are then surgically implanted into the tissue to be treated.

When the pharmaceutical composition of the present invention is used for practical treatment, the dosage of the polypeptide of the present invention or its pharmaceutically acceptable salt as an active ingredient can be determined reasonably according to the body weight, age, sex, and degree of symptoms of each patient to be treated.

The chimeric T cell receptors of the invention are useful as pharmaceuticals or diagnostic agents. May be modified or otherwise improved to obtain characteristics more suitable for use as a pharmaceutical or diagnostic agent.

In some embodiments, there is provided a nucleic acid library comprising sequences encoding a plurality of chimeric T cell receptors according to any one of the chimeric T cell receptors described above.

In some embodiments, there is provided a method of screening a library of nucleic acids according to any one of the embodiments described above for sequences encoding chimeric T cell receptors specific for a target antigen comprising: a) introducing the nucleic acid library into a plurality of cells such that the chimeric T cell receptor is expressed on the surface of the plurality of cells; b) incubating a plurality of cells with a ligand comprising a target antigen or one or more epitopes contained therein; c) collecting cells bound to the ligand; and d) isolating sequences encoding the chimeric T cell receptor from the cells collected in step c), thereby identifying the chimeric T cell receptor specific for the target antigen.

Also provided are methods of manufacture, articles of manufacture, and kits suitable for any of the constructs described herein.

In some embodiments, there is also provided a use of a chimeric T cell receptor of any one of the above chimeric T cell receptors in the preparation of a kit for treating or diagnosing a target antigen-associated disease in an individual in need thereof.

In some embodiments, there is provided a method of killing a target cell presenting a target antigen, comprising contacting the target cell with an effector cell expressing a chimeric T cell receptor according to any one of the chimeric T cell receptors described above, wherein the chimeric T cell receptor specifically binds to the target antigen.

In some embodiments, a method of killing a target cell presenting a target antigen is provided, wherein the chimeric T cell receptor specifically binds to the target antigen. Wherein the antibody heavy chain variable region and light chain variable region specifically bind to the antigen binding moiety of the target antigen.

In some embodiments, the contacting is in vivo according to any of the target cell killing methods described above. In some embodiments, the contacting is in vitro.

In some embodiments, there is provided a method of treating a target antigen associated disease in an individual in need thereof comprising administering to the individual an effective amount of a pharmaceutical composition comprising a chimeric T cell receptor according to the above or the effector cell.

In some embodiments, there is provided a method of treating a target antigen-associated disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising an effector T cell comprising a chimeric T cell receptor that specifically binds to a target antigen, comprising: a) a first polypeptide fused to an antibody heavy chain variable region; b) a second polypeptide fused to the variable region of the antibody light chain; wherein the antibody heavy chain variable region and light chain variable region specifically bind to the antigen binding moiety of the target antigen. Preferably, the chimeric T cell receptor is any of the chimeric T cell receptors described above.

In some embodiments, there is provided a method of treating a T cell mediated disorder arising from cell, tissue, body part or organ transplantation in a subject in need thereof, comprising the steps of: transplanting a cell, tissue, body part, or organ into the subject; and administering two or more doses of a pharmaceutically acceptable amount of any one of the foregoing chimeric T cell receptors to the subject.

In some embodiments, the disease is cancer according to any one of the methods above. In some embodiments, the cancer is selected from the group consisting of: adrenocortical carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, bile duct carcinoma, colorectal carcinoma, esophageal carcinoma, glioblastoma, glioma, hepatocellular carcinoma, head and neck carcinoma, renal carcinoma, lymphoma, leukemia, lung carcinoma, melanoma, mesothelioma, multiple myeloma, pancreatic carcinoma, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian carcinoma, prostate carcinoma, sarcoma, gastric carcinoma, uterine carcinoma and thyroid carcinoma. In some embodiments, the target antigen-associated disease is a viral infection. In some embodiments, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Kaposi's sarcoma associated herpes Virus (KSHV), Human Papilloma Virus (HPV), Molluscum Contagiosum Virus (MCV), human T-cell leukemia Virus 1(HTLV-1), HIV (human immunodeficiency Virus), and Hepatitis C Virus (HCV).

More preferably, the disease is adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, renal cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer, thyroid cancer, and the like.

In some embodiments, there is provided a method of treating a target antigen associated disease in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition comprising a nucleic acid encoding a chimeric T cell receptor according to any one of the chimeric T cell receptors described above.

The polynucleotides of the present invention may be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA techniques. Generally, the following steps are performed: (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a chimeric T cell receptor polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide; (2) culturing the host cell in a suitable medium; (3) the chimeric T cell receptor polypeptides of the invention are isolated and purified from the culture medium or cells.

In some embodiments, the present invention provides a use of the chimeric T cell receptor, the complex, the nucleic acid, the vector or the effector cell of any one of the preceding claims in the preparation of a kit, preparation or pharmaceutical composition for treating or diagnosing a target antigen-associated disease in a subject in need thereof.

In some embodiments, the present invention provides a method of treating a target antigen-associated disease or cancer or a disease associated with a viral infection in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a chimeric T cell receptor, the complex, nucleic acid, vector, or effector cell of any of the foregoing.

In particular, the cancer is selected from the group consisting of: adrenocortical, bladder, breast, cervical, biliary, colorectal, esophageal, glioblastoma, glioma, hepatocellular, head and neck, renal, lymphoma, leukemia, lung, melanoma, mesothelioma, multiple myeloma, pancreatic, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian, prostate, sarcoma, gastric, uterine and thyroid cancers; alternatively, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Kaposi's sarcoma-associated herpes Virus (KSHV), Human Papilloma Virus (HPV), Molluscum Contagiosum Virus (MCV), human T-cell leukemia Virus 1(HTLV-1), HIV (human immunodeficiency Virus), and Hepatitis C Virus (HCV).

Classical CAR molecules consist of a single-chain antibody region, a hinge region, a transmembrane region, and an intracellular signal region, which can be presented on the surface of a T cell. The single chain antibody region comprises the heavy chain variable region and the light chain variable region of an antibody (in some cases, the IgG CH1 region is also included to play a structural role), and the two regions are connected through a flexible connecting peptide. The intracellular signaling region is composed of co-stimulatory signaling molecules (4-1BB, CD28, etc.) and signaling molecule CD3 zeta in tandem.

The chimeric T cell receptor constructed by the application is connected by two polypeptide chains through a disulfide bond covalent bond after being expressed in cells, wherein the first polypeptide is an antibody heavy chain variable region (V)_H) Fused to the TCR α constant region (C α), and the second polypeptide is an antibody light chain variable region (V)_L) And the TCR β constant region (C)_β) Fusing to obtain the product; the chimeric T cell receptor functions as a complex with the endogenous CD3 subunits (epsilon, delta, lambda, zeta) expressed by T cells. The gene sequence of the chimeric T cell receptor is connected through furin and a p2A protease cutting site polypeptide segment, the two polypeptide chains are transcribed and translated together to express a protein, and then the protein is cut into two independent proteins by furin and a protease corresponding to p 2A. Chimeric T cell receptors can be found in a variety of combinations: antibody heavy chain variable region (V)_H) Variable region of antibody light chain (V) fused to TCR α constant region (C α)_L) And the TCR β constant region (C)_β) Fusion, or antibody light chain variable region (V)_L) Variable region of antibody heavy chain (V) fused to TCR α constant region (C α)_H) And the TCR β constant region (C)_β) Fusion, and exchange of both sequences relative to furin and P2A the light and heavy chain variable regions of the antibody can be replaced with antibody variable regions of various specificities, such as anti-EGFR, CD19, CD20, etc. variants of the TCR α and β constant regions can also exist, including wild-type TCR α and β constant regions, cysteine single-point mutant TCR α and β constant regions, human murine chimeric TCR α and β constant regions, and human murine chimeric TCR α and β constant regions containing cysteine single-point mutations.

The invention obtains the T cell receptor with high stability and high specificity, and can be used for diagnosing and treating diseases. The inventors have innovatively adopted a number of strategies in the TCR design process. The present application is based on the engineering of disulfide bonds introduced into the constant region of the TCR molecule. There are theoretically many sites in the TCR at which the artificial interchain disulfide bond can be formed, but finding an appropriate site in the TCR at which the artificial interchain disulfide bond can be formed makes it very difficult for a TCR containing the artificial interchain disulfide bond to be able to be successfully renatured, refolded to give high yield, high stability, and having specific binding activity to its protoligand. Those skilled in the art have endeavored to develop TCRs containing artificial interchain disulfide bonds that are well renatured, refolded, purified, and have high stability and high renaturation yield while specifically binding to their proto-ligand. The invention can reduce the new reactivity of TCR-T cells and reduce the mismatching by modifying the disulfide bond at a specific site.

The invention introduces an antibody antigen binding fragment to be fused with an original TCR to form an antibody-T cell receptor-bound chimeric T cell receptor (STAR), further improves the pairing of α chain and β chain, improves the binding activity of the TCR, more importantly, has less modification to the original TCR in vivo compared with CAR-T, reduces the introduction of exogenous amino acids, reduces the occurrence risk of side effects and improves the safety.

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. The present invention will be described in further detail with reference to examples.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

the STAR is expressed in cells and then is connected together through a disulfide bond covalent bond, wherein the first polypeptide chain is formed by fusing an antibody heavy chain variable region (VH) and a TCR α constant region (C α), the second polypeptide chain is formed by fusing an antibody light chain variable region (VL) and a TCR β constant region (C β), and the STAR can form a complex with CD3 subunits (epsilon, delta, lambda and zeta) endogenously expressed by T cells to play a function.

FIG. 2 is a schematic diagram of the gene structure sequence of STAR and CAR molecule, the STAR gene sequence is connected through polypeptide segments of furin and P2A protease cleavage sites, the two polypeptide chains are transcribed and translated together to form a protein, and then cleaved by furin and a protease corresponding to P2A to form two independent proteins, STAR can be combined in various ways, i.e., an antibody heavy chain variable region (VH) is fused with a TCR α constant region (C α), an antibody light chain variable region (VL) is fused with a TCR β constant region (C β), or an antibody light chain variable region (VL) is fused with a TCR α constant region (C α), an antibody heavy chain variable region (VH) is fused with a TCR β constant region (C β), and the sequences are exchanged with respect to furin and P2A.

FIG. 3 Membrane-Loading of EGFR-targeting STAR in human T cells the use of lentiviral vectors introduced the STAR gene into the human T cell line Jurkat Clone 5 (endogenous TCR-deleted Jurkat subclone) 3 days after infection, flow antibody staining with anti-human TCR- α/β was followed by flow detection.

Figure 4 ability of STAR targeting EGFR to bind antigen at the surface of human T cell membrane. Jurkat Clone 5 cells 3 days after the introduction of the STAR gene were stained with flow-through antibodies against the antigen proteins EGFR-His and anti-His-APC, and then subjected to flow-through detection. It was found that STAR showed stronger staining compared to the negative control cells of native E1-TCR (specificity not directed against EGFR) and the staining level was comparable to that of the CAR of anti-EGFR.

Figure 5 ability of STAR-mediated T cell activation targeting EGFR. JurkatClone 5 cells 3 days after the introduction of the STAR gene were cultured in an EGFR antigen-coated cell culture plate and co-incubated with tumor cells A549 (EGFR-positive human lung cancer cell line), respectively. After 24 hours, cells were harvested and stained with flow antibody against anti-human CD69-FITC prior to flow detection. Positive on the abscissa CD69 are cells expressing the T cell activation marker CD69 molecule. STAR can be found to cause T cells to express an activation marker of CD69 upon antigen stimulation, i.e., STAR can mediate T cell activation following antigen stimulation and to a comparable extent to CAR. It was also found that in the resting state without antigenic stimulation, STAR has no self-activation phenomenon, whereas CAR has a higher level of self-activation.

Figure 6 function of EGFR-targeted STAR in human primary T cells. Human peripheral blood cells were taken and CD4+ and CD8+ T cells were purified therefrom using pan T cell isolation kit. The STAR gene was then transferred into T cells using lentiviral vectors 72 hours after the T cells were activated by stimulation with anti-CD 3/CD28 antibodies. After viral infection, the cells were cultured in RPMI 1640 medium containing 20% serum and 200IU/mLIL-2 to a sufficient amount. T cells were co-cultured with A431 cells, a highly EGFR positive human skin cancer cell, and T cell activation and target cell death were examined. After 8 hours of co-culture, T cells were stained and both STAR and CAR were found to mediate T cell activation. STARs can cause significant T cell activation, as seen at the levels of the T cell marker CD69 (fig. 6a) and the T cell cytokine IFN- γ (fig. 6 b). After 24 hours of co-incubation, cell supernatants were taken to measure Lactate Dehydrogenase (LDH) levels (FIG. 6c), which reflects target cell death. The results show that both STAR-T and CAR-T cells have obvious killing effect on target cells.

Figure 7 ability of STAR targeting CD19 to mediate T cell activation. The STAR gene targeting CD19 was introduced into the human T cell line Jurkat Clone 5 using a lentiviral vector. T cells 3 days after infection were co-incubated with Raji, Mino, LY-1 tumor cells (CD19 and CD20 positive human lymphoma cell lines), respectively. After 24 hours, cells were harvested and stained with flow antibody against anti-humanCD69-FITC prior to flow detection. Positive on the ordinate CD69 were cells expressing the T cell activation marker CD69 molecule. STAR can be found to cause T cells to express an activation marker of CD69 upon antigen stimulation, i.e., STAR can mediate T cell activation following antigen stimulation and to a comparable extent to CAR. It was also found that in the resting state without antigenic stimulation, STAR has no self-activation phenomenon, whereas CAR has a higher level of self-activation.

Figure 8 function of STAR targeting CD19 in human primary T cells. Human peripheral blood cells were taken and CD4+ and CD8+ T cells were purified therefrom using pan T cell isolation kit. The STAR gene targeting CD19 was then transferred into T cells with lentiviral vectors 72 hours after activation of the T cells by stimulation with anti-CD 3/CD28 antibodies. After viral infection, the cells were cultured in RPMI 1640 medium containing 20% serum and 200IU/mL IL-2 to a sufficient amount. T cells were co-cultured with Raji and LY-1 cells and target cell death was detected. After 8 hours of co-culture, T cells were stained and both STAR and CAR were found to mediate T cell activation. At the level of T cell cytokine IFN- γ (fig. 4), STAR can cause significant T cell activation (at levels higher than CAR). The expression level result of IFN-gamma in T cells shows that target cells have obvious activation effect on STAR-T cells.

Figure 9 ability of STAR targeting CD20 to mediate T cell activation. The STAR gene targeting CD20 was introduced into the human T cell line Jurkat Clone 5 using a lentiviral vector. T cells 3 days after infection were co-incubated with Raji, Mino, LY-1 tumor cells (CD19 and CD20 positive human lymphoma cell lines), respectively. After 24 hours, cells were harvested and stained with flow antibody against anti-humanCD69-FITC prior to flow detection. Positive on the ordinate CD69 were cells expressing the T cell activation marker CD69 molecule. STAR can be found to cause T cells to express an activation marker of CD69 upon antigen stimulation, i.e., STAR can mediate T cell activation following antigen stimulation and to a comparable extent to CAR. It was also found that in the resting state without antigenic stimulation, STAR has no self-activation phenomenon, whereas CAR has a higher level of self-activation.

Figure 10 function of STAR targeting CD20 in human primary T cells. Human peripheral blood cells were taken and CD4+ and CD8+ T cells were purified therefrom using pan T cell isolation kit. The STAR gene targeting CD20 was then transferred into T cells with lentiviral vectors 72 hours after activation of the T cells by stimulation with anti-CD 3/CD28 antibodies. After viral infection, the cells were cultured in RPMI 1640 medium containing 20% serum and 200IU/mL IL-2 to a sufficient amount. T cells were co-cultured with Raji and LY-1 cells and target cell death was detected. After 8 hours of co-culture, T cells were stained and both STAR and CAR were found to mediate T cell activation. At the level of T cell cytokine IFN- γ (fig. 4), STAR can cause significant T cell activation (at levels higher than CAR). The expression level result of IFN-gamma in T cells shows that target cells have obvious activation effect on STAR-T cells.

Detailed Description

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the assays, screens, and treatments of the present invention can be made and used, and it is to be understood that these examples are intended to be illustrative only and are not to be construed as limiting the invention.

Example 1 construction of EGFR-Targeted STAR

The specific construction method comprises the following steps:

1. TCR constant region sequence determination

The constant regions (C regions) of α chain and β chain of TCR in STAR were derived from cDNA of human peripheral blood T cells by PCR molecular cloning, and 48 th and 57 th amino acid sites of the constant regions of α chain and β chain, respectively, were mutated to cysteine to facilitate formation of an additional disulfide bond between α chain and β chain based on the original TCR sequence, increasing the efficiency of pairing, and designated as E1-TCR.

2. EGFR-targeting antibody sequence determination

Cetuximab (Cetuximab) is selected as the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL), and the contents of the present invention are explained as examples, and other known antibodies can be substituted.

3. Star construction targeting EGFR

STAR contains two polypeptide chains, the Cetux-VL is fused with the TCR β chain to form a first polypeptide chain, and the Cetux-VH is fused with the α chain to form a second polypeptide chain, the gene sequence of the STAR is connected with the polypeptide segment of the protease cleavage site of furin and p2A, the two polypeptide chains are transcribed and translated together into a fused polypeptide, and then are cleaved by protease corresponding to furin and p2A to form two independent protein subunits, and the two subunits are covalently bonded through disulfide bonds and form a complex with the CD3 subunit (epsilon, delta, gamma, zeta) endogenous to T cells (as shown in figure 1 and figure 2).

The entire gene was inserted into the lentiviral expression vector pHAGE via restriction endonuclease sites NheI and NotI, which carries ampicillin resistance, the EF1 α promoter, and the IRES-RFP fluorescent reporter gene.

4. Cloning and Assembly of Gene fragments

The four fragments "Cetux VL", "TCR β -C", "Cetux-VH" and "TCR α -C" were cloned from pHAGE-Cetux-28ZCAR vector and pHAGE-E1-TCR vector, respectively, each pair of primers has 25bp bases homologous to the front and back, and the four fragments were recombinantly ligated into lentiviral vector in one step by the Gibson Assembly method to obtain STAR.

The nucleotide sequence of the E1 TCR α chain constant region cysteine mutant is SEQ ID NO 1;

the nucleotide sequence of the E1 TCR β chain constant region cysteine mutant is SEQ ID NO 2;

the nucleotide sequence of the Cetux VH is SEQ ID NO. 3;

the nucleotide sequence of the Cetux VL is SEQ ID NO. 4;

5. vector transformation and sequencing

The product of the Gibson Assembly was transformed into the DH5 α strain and allowed to grow overnight on LB plates containing ampicillin, and the monoclonal clones were selected for sequencing using seq-pHAGE-F and seq-pHAGE-R primers on pHAGE vectors.

6. Plasmid extraction

The bacteria with the correct sequencing result are inoculated in LB liquid medium and cultured overnight. The plasmid was extracted using a kit having an endotoxin removing function. The plasmid concentration is measured by Nanodrop, the final concentration of the plasmid is about 1000ng/ul, and the A260/A280 value is more than 1.8.

Example 2 validation of EGFR-Targeted STAR function

1. Lentiviral packaging

The pHAGE vector carrying the gene of interest was transfected into 293T cells (transfected with PEI) in proportion to the packaging plasmids pMD2.G and psPAX 2. Cell culture supernatants were collected for 48 and 72 hours and mixed with PEG8000, and centrifuged after standing overnight to obtain viral pellets. Resuspend with small volume of medium, effect virus concentration.

2. Lentiviral infection of human T cell lines

Lentiviruses carrying the gene of interest were infected with Jurkat clone 5 cells (endogenous TCR-deleted Jurkat subclone). The concentrated lentivirus was added to T cell culture medium along with the transfer-assisting agent Polybrene and centrifuged at 1500rpm at 32 ℃ for 2 hours. After 3 days of infection, the fluorescent reporter gene can be observed and the expression of the target protein can be detected.

3. EGFR-targeting STAR epimembranous situation and antigen binding Capacity detection

T cells 3 days after infection were taken and stained with flow antibody against anti-human TCR α/β -BV421 prior to flow detection it was found (FIG. 3) that STAR could be stained with anti-TCR α/β antibody compared to non-transgenic negative control cells and at a level comparable to native E1-TCR this result indicates that the STAR molecule could be filmed and its α and β chains could be paired.

T cells infected 3 days later were stained with flow-through antibodies against the antigen proteins EGFR-His and anti-His-APC, followed by flow-through detection. It can be found (fig. 4) that STAR shows stronger staining compared to the negative control cells of native E1-TCR (specific not against EGFR) and the staining level is comparable to CAR of anti-EGFR. This result indicates that STAR has antigen recognition and binding capacity comparable to CAR molecules.

4. Co-incubation of EGFR-targeting STAR-T cells with target cells and detection of T cell activation mediated thereby

T cells after 3 days of infection were taken, cultured in EGFR antigen-coated cell culture plates, and co-incubated with tumor cells A549 (EGFR-positive human lung cancer cell line), respectively. After 24 hours, cells were harvested and stained with flow antibody against anti-human CD69-FITC prior to flow detection (FIG. 5). Positive on the abscissa CD69 are cells expressing the T cell activation marker CD69 molecule. STAR can be found to cause T cells to express an activation marker of CD69 upon antigen stimulation, i.e., STAR can mediate T cell activation following antigen stimulation and to a comparable extent to CAR. It was also found that in the resting state without antigenic stimulation, STAR has no self-activation phenomenon, whereas CAR has a higher level of self-activation.

5. Isolation, culture and lentivirus infection of human primary T cells

Human peripheral blood cells were obtained, and CD4 and CD8T cells were purified using a whole T cell magnetic bead isolation kit. T cells are stimulated and activated in a culture dish coated with an anti-CD 3/CD28 antibody for 48-72 hours, and phenomena such as the increase of the volume, the growth of clusters and the polarization of the shapes of the T cells are observed. At this time, the target gene was transferred into T cells using a lentiviral vector by centrifugation at 1500rpm at 32 ℃ for 2 hours. After viral infection, the cells were cultured in RPMI 1640 medium containing 20% serum and 200IU IL-2 to a sufficient amount.

6. Functional validation of EGFR-targeted STAR in human primary T cells

T cells were co-cultured with A431 cells (a highly EGFR-positive human skin cancer cell) at a quantitative ratio of 1:1 to 5:1, and T cell activation and target cell death were examined. After 8 hours of co-culture, T cells were stained and both STAR and CAR were found to mediate T cell activation. STARs can cause significant T cell activation, as seen at the levels of the T cell marker CD69 (fig. 6a) and the T cell cytokine IFN- γ (fig. 6 b). After 24 hours of co-incubation, cell supernatants were taken to measure Lactate Dehydrogenase (LDH) levels (FIG. 6c), which reflects target cell death. The results show that STAR-T cells have obvious killing effect on target cells.

Example 3 construction of STAR targeting CD19

The specific construction method comprises the following steps:

1. TCR constant region sequence determination

The cDNA derived from human peripheral blood T cells or mouse spleen T cells is cloned by PCR molecule, on the basis of original TCR sequence, the 48 th and 57 th amino acid sites of the constant region of α chain and β chain are mutated into cysteine respectively to help the α chain and β chain to form an additional disulfide bond and increase the efficiency of mutual pairing, and the cDNA is named as E1-TCR (human source) or E11-TCR (mouse source), respectively.

2. Antibody sequence determination targeting CD19

The antibody heavy chain variable region (VH) and antibody light chain variable region (VL) are selected from scFv fragments of a murine monoclonal antibody (clone number FMC63) specific for CD19, which are merely examples to illustrate the content of the invention, and other known antibodies can be substituted.

3. STAR construction targeting CD19

STAR contains two polypeptide chains, FMC63-VL is fused with TCR β chain to form a first polypeptide chain, FMC63-VH is fused with FMC α chain to form a second polypeptide chain, the gene sequence of STAR is connected with polypeptide segment of protein cleavage site p2A through furin, the two polypeptide chains are transcribed and translated together to form a fused polypeptide, then are cleaved into two independent protein subunits by protease corresponding to furin and p2A, the two subunits are covalently combined through disulfide bonds and form a complex with CD3 subunit (epsilon, delta, gamma and zeta) endogenous to T cells.

4. Cloning and Assembly of Gene fragments

The four fragments "FMC 63-VL", "TCR β -C", "FMC 63-VH" and "TCR α -C" were cloned from pHAGE-FMC63-41BBzCAR vector and pHAGE-E1-TCR vector respectively, each pair of primers has 25bp bases homologous to the front and back, and the four fragments were further recombined and ligated into lentiviral vector by Gibson Assembly method to obtain STAR.

the nucleotide sequence of FMC63-VH is SEQ ID NO. 5;

the nucleotide sequence of FMC63-VL is SEQ ID NO 6;

5. vector transformation and sequencing

6. Plasmid extraction

Example 4 functional validation of STAR targeting CD19

1. Lentiviral packaging

2. Lentiviral infection of human T cell lines

Co-incubation of FMC63-STAR-T cells with target cells and detection of their ability to mediate T cell activation

T cells 3 days after infection were co-incubated with Raji, Mino, LY-1 tumor cells (CD19 and CD20 positive human lymphoma cell lines), respectively. After 24 hours, cells were harvested and stained with flow antibody against anti-human CD69-FITC prior to flow detection (FIG. 7). Positive on the ordinate CD69 were cells expressing the T cell activation marker CD69 molecule. STAR can be found to cause T cells to express an activation marker of CD69 upon antigen stimulation, i.e., STAR can mediate T cell activation following antigen stimulation and to a comparable extent to CAR. It was also found that in the resting state without antigenic stimulation, STAR has no self-activation phenomenon, whereas CAR has a higher level of self-activation.

4. Isolation, culture and lentivirus infection of human primary T cells

Human peripheral blood cells were obtained, and CD3 was isolated from the cells using a whole T cell magnetic bead isolation kit⁺T cells were purified. T cells are stimulated and activated in a culture dish coated with an anti-CD 3/CD28 antibody for 48-72 hours, and phenomena such as the increase of the volume, the growth of clusters and the polarization of the shapes of the T cells are observed. At this time, the target gene was transferred into T cells using a lentiviral vector by centrifugation at 1500rpm at 32 ℃ for 2 hours. After viral infection, the cells were cultured in RPMI 1640 medium containing 20% serum and 200IU/mL IL-2 to a sufficient amount.

5. Functional validation of CD 19-targeted STAR in human primary T cells

T cells were co-cultured with Raji and LY-1 cells at a ratio of 1:1 to 5:1, and T cell activation and target cell death were examined. After 8 hours of co-culture, T cells were stained and both STAR and CAR were found to mediate T cell activation. At the level of the T cell cytokine IFN- γ (fig. 8), STAR can cause significant T cell activation. The expression level result of IFN-gamma in T cells shows that target cells have obvious activation effect on STAR-T cells.

Example 5 construction of STAR targeting CD20

The specific construction method comprises the following steps:

1. antibody sequence determination targeting CD20

The antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) are selected from scFv fragments of the CD 20-specific antibody Ofatumumab (Ofatumumab, OFA), which are merely examples to illustrate the content of the present invention, and other known antibodies may be substituted.

2. STAR construction targeting CD20

STAR contains two polypeptide chains, wherein OFA-VL and TCR β chain are fused into a first polypeptide segment, OFA-VH and α chain are fused into a second polypeptide segment, the gene sequence of STAR is connected through furin and p2A protease cleavage site polypeptide segments, the two polypeptide chains are transcribed and translated into a fused polypeptide together, and then are cleaved into two independent protein subunits by furin and p2A corresponding protease, the two protein subunits are covalently bonded through disulfide bonds and form a complex with CD3 subunits (epsilon, delta, gamma and zeta) endogenous to T cells.

3. Cloning and Assembly of Gene fragments

The four fragments "OFA-VL", "TCR β -C", "OFA-VH" and "TCR α -C" were obtained by cloning from pHAGE-OFA-41BBzCAR vector and pHAGE-E11-TCR vector, respectively, each pair of primers has 25bp bases homologous to the former and latter, and the four fragments were recombinantly ligated into lentiviral vector in one step by the Gibson Assembly method, thereby obtaining STAR.

The nucleotide sequence of the E11 TCR α chain constant region cysteine mutant is SEQ ID NO. 7;

the nucleotide sequence of the E11 TCR β chain constant region cysteine mutant is SEQ ID NO 8;

the nucleotide sequence of the OFA-VH is SEQ ID NO. 9;

the nucleotide sequence of the OFA-VL is SEQ ID NO. 10;

4. vector transformation and sequencing

5. Plasmid extraction

Example 6 functional validation of STAR targeting CD20

1. Lentiviral packaging

2. Lentiviral infection of human T cell lines

Co-incubation of OFA-STAR-T cells with target cells and detection of T cell activation mediated thereby

T cells 3 days after infection were co-incubated with Raji, Mino, LY-1 tumor cells (CD19 and CD20 positive human lymphoma cell lines), respectively. After 24 hours, cells were harvested and stained with flow antibody against anti-human CD69-FITC prior to flow detection (FIG. 9). Positive on the ordinate CD69 were cells expressing the T cell activation marker CD69 molecule. STAR can be found to cause T cells to express an activation marker of CD69 upon antigen stimulation, i.e., STAR can mediate T cell activation following antigen stimulation and to a comparable extent to CAR. It was also found that in the resting state without antigenic stimulation, STAR has no self-activation phenomenon, whereas CAR has a higher level of self-activation.

4. Isolation, culture and lentivirus infection of human primary T cells

5. Functional validation of CD 20-targeted STAR in human primary T cells

T cells were co-cultured with Raji and LY-1 cells at a ratio of 1:1 to 5:1, and T cell activation and target cell death were examined. After 8 hours of co-culture, T cells were stained and both STAR and CAR were found to mediate T cell activation. At the level of the T cell cytokine IFN- γ (fig. 10), STAR can cause significant T cell activation. The expression level result of IFN-gamma in T cells shows that target cells have obvious activation effect on STAR-T cells.

Therefore, the invention has successfully constructed STAR of multiple targets, successfully verified that the antibody-T cell chimeric receptor involved in the application can form a complex with CD3 subunits (epsilon, delta, lambda, zeta) endogenously expressed by T cells to function, can mediate activation of T cells after antigen stimulation, and has comparable antigen activation degree compared with the corresponding antibody-Chimeric Antigen Receptor (CAR) prepared, and more importantly, STAR has no self-activation phenomenon and CAR has high self-activation phenomenon in a resting state without antigen stimulation. For purposes of brevity, the present invention is a list of exemplary STARs that are sufficient to support the successful construction and the outstanding technical effects of the STARs of the present invention.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A chimeric T cell receptor (STAR) that specifically binds to a target antigen, the chimeric T cell receptor comprising:

2. The chimeric T-cell receptor of claim 1, wherein:

(1) when the first subunit of the T cell receptor is a α chain, the second subunit of the T cell receptor is a β chain, or,

(2) when the first subunit of the T cell receptor is a β chain, the second subunit of the T cell receptor is a α chain, or,

(3) when the first subunit of the T cell receptor is a gamma chain, the second subunit of the T cell receptor is a delta chain; or the like, or, alternatively,

(4) when the first subunit of the T cell receptor is a delta chain, the second subunit of the T cell receptor is a gamma chain.

3. The chimeric T-cell receptor according to any one of claims 1-2, wherein said first and second peptide chains are bound by disulfide bonds upon expression in a T-cell.

4. The chimeric T-cell receptor according to any one of claims 1-3, wherein: the species source of the T cell receptor first subunit constant region and the T cell receptor second subunit constant region is human or mouse, and comprises different protein subtypes.

5. The chimeric T-cell receptor according to any one of claims 1-4, wherein: the chimeric T cell receptor is subjected to amino acid sequence modification to reduce mismatching with an endogenously expressed T cell receptor, wherein the modification comprises but is not limited to amino acid point mutation modification and polypeptide fragment replacement modification.

6. The chimeric T-cell receptor according to any one of claims 1-5, wherein:

(1) the first subunit of the T cell receptor is a TCR α chain, the 48 th amino acid of the constant region of which is mutated into cysteine, and the second subunit of the T cell receptor is a TCR β chain, the 57 th amino acid of the constant region of which is mutated into cysteine, or

(2) The first subunit of the T cell receptor is a TCR α chain, the 85 th amino acid of the constant region of the T cell receptor is mutated into alanine, and the second subunit of the T cell receptor is a TCR β chain, and the 88 th amino acid of the constant region is mutated into glycine.

7. The chimeric T-cell receptor according to any preceding claim, wherein the target antigen is a tumor-specific antigen or a virus-specific antigen.

8. The chimeric T-cell receptor according to any one of claims 1 to 7, wherein said target antigen is selected from the group consisting of CD19, CD20, EGFR, Her2, PSCA, CD123, CEA (carcinoembryonic antigen), FAP, CD133, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, VEGFR, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, lewis y, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, CD 171.

9. The chimeric T cell receptor according to any preceding claim, wherein the antibody, antibody heavy chain variable region or antibody light chain variable region is derived from IMCC225 (Cetuximab, Cetuximab/Cetux), Ofatumumab (Ofatumumab), CD19 monoclonal antibody FMC63, rituximab (rituximab), Avastin (bevacizumab), BEC2 (adoumumab), Bexxar (tositumomab), Campath (alemtuzumab), Herceptin (trastuzumab), lymphocid (epratuzumab), MDX-210, Mylotarg (gemuzumab ozomicin), mab 17-1A (epratuzumab), theragyn (pemumomab), Zamyl, Zevalin (ibritumomab tiuj) or a high affinity antibody obtained by screening.

10. The chimeric T-cell receptor according to any preceding claim, wherein the target antigen-associated disease is cancer or a disease associated with a viral infection.

11. The chimeric T cell receptor according to claim 10, wherein the cancer is selected from the group consisting of: adrenocortical, bladder, breast, cervical, biliary, colorectal, esophageal, glioblastoma, glioma, hepatocellular, head and neck, renal, leukemia, lymphoma, lung, melanoma, mesothelioma, multiple myeloma, pancreatic, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian, prostate, sarcoma, gastric, uterine and thyroid cancers; or, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr virus (EBV), Hepatitis B Virus (HBV), Kaposi's sarcoma-associated herpes virus (KSHV), Human Papilloma Virus (HPV), Molluscum Contagiosum Virus (MCV), human T-cell leukemia virus 1(HTLV-1), HIV (human immunodeficiency virus), and Hepatitis C Virus (HCV).

12. The chimeric T-cell receptor according to any preceding claim, wherein said first and second peptidic chains form a complex with the endogenous CD3 subunits (epsilon, delta, gamma, zeta) of the T-cell.

13. A complex formed by a chimeric T cell receptor that specifically binds to a target antigen, wherein the chimeric T cell receptor of any preceding claim forms a complex with a T cell endogenously expressed CD3 subunit (epsilon, delta, gamma, zeta) and is capable of mediating a T cell-associated signaling pathway upon activation by the target antigen.

14. A nucleic acid encoding the chimeric T-cell receptor according to any one of claims 1 to 12 or the first and second peptide chains in the complex according to claim 13.

15. A nucleic acid, comprising:

(3) sequentially comprises an antibody light chain variable region, a T Cell Receptor (TCR) α chain constant extracellular section, a transmembrane region and an intracellular tail end, a linker, an antibody heavy chain variable region, a T Cell Receptor (TCR) β chain constant extracellular section, a transmembrane region and an intracellular tail end, or,

16. A vector comprising a nucleic acid sequence encoding the chimeric T-cell receptor according to any one of claims 1 to 12 or the first and second peptide chains in the complex according to claim 13, or comprising the nucleic acid according to claims 14 to 15.

17. The vector of claim 16, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

18. An effector cell expressing a chimeric T-cell receptor according to any one of claims 1 to 12 or a complex according to claim 13 on its cell surface.

19. The effector cell according to claim 18, wherein the effector cell is a T cell, preferably wherein the effector cell is a cytotoxic T cell, a helper T cell, a natural killer T cell, or an suppressor T cell.

20. A pharmaceutical composition comprising the chimeric T cell receptor of any one of claims 1-12, the complex of claim 13, the nucleic acid of any one of claims 14-15, the vector of any one of claims 16-17, or the effector cell of any one of claims 18-19, and a pharmaceutically acceptable carrier.

21. A kit comprising the chimeric T cell receptor of any one of claims 1-12, the complex of claim 13, the nucleic acid of any one of claims 14-15, the vector of any one of claims 16-17 or the effector cell of any one of claims 18-19, the pharmaceutical composition of claim 20.

22. Use of a chimeric T cell receptor according to any one of claims 1 to 12, a complex according to claim 13, a nucleic acid according to any one of claims 14 to 15, a vector according to any one of claims 16 to 17 or an effector cell according to any one of claims 18 to 19 for the preparation of a kit, preparation or pharmaceutical composition for the treatment or diagnosis of a target antigen-associated disease in a subject in need thereof.

23. A method of killing a target cell that presents a target antigen, comprising contacting the target cell with the effector cell of any one of claims 18-19, wherein the chimeric T cell receptor specifically binds to the target antigen.

24. A method of treating a target antigen-associated disease or a cancer or a virus infection-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the chimeric T cell receptor of any one of claims 1-12, the complex of claim 13, the nucleic acid of any one of claims 14-15, the vector of any one of claims 16-17, or the effector cell of any one of claims 18-19, the pharmaceutical composition of claim 20.

25. The method of claim 24, wherein the cancer is selected from the group consisting of: adrenocortical, bladder, breast, cervical, biliary, colorectal, esophageal, glioblastoma, glioma, hepatocellular, head and neck, renal, lymphoma, leukemia, lung, melanoma, mesothelioma, multiple myeloma, pancreatic, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian, prostate, sarcoma, gastric, uterine and thyroid cancers; alternatively, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr virus (EBV), Hepatitis B Virus (HBV), Kaposi's sarcoma-associated herpes virus (KSHV), Human Papilloma Virus (HPV), Molluscum Contagiosum Virus (MCV), human T-cell leukemia virus 1(HTLV-1), HIV (human immunodeficiency virus), and Hepatitis C Virus (HCV).