CN110818802A - A chimeric T cell receptor STAR and its application - Google Patents

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

A chimeric T cell receptor STAR and its application

technical field

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

Background technique

Chimeric antigen receptor T-cell therapy (CAR-T) and T-cell receptor therapy (TCR-T) are two cutting-edge gene therapies that utilize a patient's own T lymphocytes to treat cancer. They can express specific receptors, target and recognize specific cells such as tumor cells, and have received extensive attention and research, from the initial basic immune research to clinical application. Based on the latest advances in synthetic biology, immunology and genetic engineering technology, it is possible to synthesize engineered T cells with enhanced specific functions.

CAR-T uses antibody fragments that can bind to specific antigens to recognize antigens on the surface of tumor cells. 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 T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a wider range of targets relative to the native T cell surface receptor TCR. At present, CAR-T technology has significant curative effect in the treatment of acute leukemia and non-Hodgkin lymphoma, and is considered to be one of the most promising tumor treatment methods.

Different from CAR-T, TCR (T cell receptor) is a molecule that specifically recognizes antigens and mediates immune responses on the surface of T cells. TCR mainly recognizes antigenic molecule polypeptides presented by histocompatibility complex molecules. In recent years, due to the great clinical success of CAR-T cell therapy, the research on TCR therapy has been relatively less interesting. However, in the treatment of solid tumors, TCR-T has obvious advantages compared with CAR-T, because the target antigen of CAR-T cell therapy is cell surface proteins, and TCR-T recognizes MHC molecules, which can Presents peptide chains obtained from cell surface and intracellular proteins, thus enabling TCR-T to target a wider variety of antigens.

It is well known that most tumor-associated antigens (TAAs) are self-antigens. Due to the selection mechanism and tolerance mechanism of the thymus, the affinity of most T cell receptors (TCRs) for T lymphocytes produced by the body against these antigens is relatively low. Limit its tumor recognition and killing effect. The cloned TCR (or chimeric receptor) that recognizes TAA with high affinity is transferred to T lymphocytes by transgenic technology, so that the redirected T cells without tumor recognition ability can effectively recognize and kill tumor cells in vitro and in vivo. TCR is a characteristic marker on the surface of all T cells, and it binds to CD3 (ε, δ, γ, ζ) non-covalently to form a TCR-CD3 complex.

TCR is a heterodimer composed of two different peptide chains. It consists of two peptide chains, α and β. Each peptide chain can be divided into two parts: variable region (V region) and constant region (C region). The constant region includes three parts: extracellular region, transmembrane region and intracellular terminal; its characteristic is that the intracellular region is very short. TCR molecules belong to the immunoglobulin superfamily, and their antigenic specificity exists in the V region. TCRs are divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, γ and δ, and TCR2 is composed of two chains, α and β. In peripheral blood, 90%-95% of T cells express TCR2; and any T cell expresses only one of TCR2 or TCR1.

Naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region, so obtaining a highly stable TCR that retains the ability to specifically bind to its original ligand (i.e. pMHC) is an important step for expressing a soluble TCR in bacteria. a very difficult thing, as described in patent document WO99/18129. Some literatures describe truncated forms of TCRs, which contain only the extracellular domain or only the extracellular and cytoplasmic domains. Although such TCRs can be recognized by TCR-specific antibodies, the yields are very low, and at low concentrations they are not The identification of the major histocompatibility complex-peptide complex indicates that it is easily denatured and not stable enough.

The advantages of TCR-T technology are: traditional adoptive immunotherapy only increases the number of effector cells, but does not improve the specificity of effector cells, and even if effector cells can bind to tumor cells, their affinity is relatively low. TCR-T technology directly transforms the "probe"-TCR of T cells that binds tumor antigens, which strengthens the specific recognition process of T cells against tumor cells, and improves the affinity of T lymphocytes for tumor cells, so that no tumor can be recognized. Competent T cells efficiently recognize and kill tumor cells in vitro and in vivo. In a word, TCR-T cell therapy increases the number of T lymphocytes while improving the killing ability of T lymphocytes to tumor cells, thereby achieving a good tumor treatment effect.

In the existing TCR-T therapy, it is generally necessary to isolate the endogenous TCR, engineer it, introduce it into brand-new T cells, and infuse them back into the human body. The number of T cells with the ability to target cancer cells will be large. It is expected to identify and attack a variety of solid tumors and hematological tumors. However, the exogenous TCR α and β chains and the endogenous α and β chains will recognize and pair with each other to form a hybrid TCR. This TCR mismatch phenomenon is considered to be widespread in TCR-T therapy, which may lead to a decrease in the expression of TCR on the cell surface and a decrease in cell activity. Mismatched TCRs may also form a new TCR of unknown specificity that could lead to autoimmune (on-target) or cross-reactive (off-target) toxicity in TCR-T. Although there is no clinical evidence for autoreactive diseases caused by TCR mismatches, the autoimmune risk brought by heterozygous TCRs cannot be ignored. Therefore, the expression and paired assembly of TCR in T cells and the affinity with pMHC are the key factors that affect the full anti-tumor ability of TCR gene therapy.

It can be seen that how to modify the TCR gene so that the TCRα chain and the β chain can be correctly paired on the surface of T cells, and their expression efficiency and affinity are enhanced, while avoiding side effects and improving safety have become one of the hot spots of TCR gene therapy in recent years. The main strategies include improving the affinity of TCRs in T cells, optimizing the pairing of TCR chains, and enhancing their surface expression efficiency. For example, some researchers have adopted the following methods to reduce TCR mismatches: introducing disulfide bonds in the TCR chain; replacing the C region of human TCR molecules with a mouse-derived conservative C region; The amino acid residue inversion of the aβTCR gene; the construction of a single-chain TCR (scTCR) chimera; the fusion of the C region of the TCR chain with the CD3 molecule; the transfer of the aβTCR gene into γδT cells, etc., but the above effects are not satisfactory. It can be seen that although the modification of TCR-T to optimize the pairing strategy of exogenous TCR molecules has been proposed, there are still many areas for improvement.

SUMMARY OF THE INVENTION

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

a) a first peptide chain obtained by fusing the variable region of the antibody heavy chain with the constant region of the first subunit of the T cell receptor (TCR); and,

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

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

In some embodiments, according to the aforementioned chimeric T cell receptor, (1) when the first subunit of the T cell receptor is an alpha chain, the second subunit of the T cell receptor is a beta 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 an α chain; or, (3) when the first subunit of the T cell receptor is When it is a gamma chain, the second subunit of the T cell receptor is a delta chain; or, (4) when the first subunit of the T cell receptor is a delta chain, the second subunit of the T cell receptor is a delta chain for the gamma chain.

Specifically, the first peptide chain and the second peptide chain are combined through a disulfide bond after being expressed in T cells.

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 and include different protein isoforms.

In some embodiments, the chimeric T cell receptor can be modified in any amino acid sequence, including but not limited to amino acid point mutation modifications, polypeptide fragment substitution modifications, to reduce mismatches with endogenously expressed T cell receptors . For example: TCRα chain, the 48th amino acid of the constant region is mutated to cysteine, and the 57th amino acid of its constant region is mutated to cysteine, so that the passage between the first polypeptide and the second polypeptide Disulfide bond connection; another example: TCRα chain, the 85th amino acid of its constant region is mutated to alanine, and the 88th amino acid of its constant region is mutated to glycine;

Specifically, (1) the first subunit of the T cell receptor is a TCRα chain, and the 48th amino acid in the constant region is mutated to cysteine, and/or, the second subunit of the T cell receptor is TCRβ or (2) the first subunit of the T cell receptor is a TCRα chain, and the amino acid at position 85 of its constant region is mutated to alanine, and/or , the second subunit of the T cell receptor is the TCRβ chain, and the 88th amino acid in the constant region is mutated to glycine.

In some embodiments, the target antigen is a tumor-specific antigen or a virus-specific antigen. The specific target antigen is selected from 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, CD171. Preferably, the antigen can be CD19, CD20, EGFR, Her2.

Specifically, in some embodiments, the antibody, antibody heavy chain variable region or antibody light chain variable region are from IMCC225 (cetuximab, Cetuximab/Cetux), rituximab (rituximab), Ofatumumab (OFA, ofatumumab), CD19 monoclonal antibody (FMC63), Avastin (bevacizumab), BEC2 (atumumab), Bexxar (tositumumab), Campath (alemtuzumab) anti), Herceptin (trastuzumab), LymphoCide (epratuzumab), MDX-210, Mylotarg (gemtuzumab ozogamicin), mAb 17-1A (edrolizumab), Theragyn ( pemtumomab), Zamyl, Zevalin (tiimtumomab) or high-affinity antibodies obtained by screening. Preferably, the antibody comprises an antigen-binding fragment selected from the group consisting of Fab, F(ab')2, Fab', scFv, Fv, VH, and VL.

In some embodiments, the target antigen-related disease is a cancer or viral infection-related disease. Specifically, the cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, Head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, Sarcoma, gastric cancer, uterine cancer and thyroid cancer; or, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-BarrVirus (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) virus) and hepatitis C virus (HCV).

In the chimeric T cell receptor of the present invention, the first polypeptide and the second polypeptide form a complex with CD3 subunits (ε, δ, γ, ζ) endogenous to T cells.

In some embodiments, the antibody heavy and light chain variable regions are derived from IMCC225 (Cetuximab, Cetuximab/Cetux), Rituximab (Rituximab), Ofatumumab (Ofatumumab) VH and VL of anti), CD19 monoclonal antibody FMC63.

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

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

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

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

Further preferably, the VH and VL derived from IMCC225 (Cetuximab, Cetuximab/Cetux), Rituximab (Rituximab), Ofatumumab (Ofatumumab), and CD19 monoclonal antibody FMC63 are respectively The TCRα chain constant region or the β chain constant region is fused to obtain a VH-TCRα chain constant region fusion or a VL-TCRβ chain constant region fusion.

Preferably, the two different fusions are connected by a furin-p2A segment peptide sequence; preferably, the two different fusions are covalently bound by disulfide bonds after being expressed in T cells. Furthermore, the two different fusions form complexes with the endogenous CD3 subunits (ε, δ, γ, ζ) of T cells.

In addition, the present invention also provides a complex formed by a chimeric T cell receptor that specifically binds to a target antigen, comprising any of the aforementioned chimeric T cell receptors and the CD3 subunit ( ε, δ, γ, ζ) form a complex and can mediate T cell-related signal transduction pathways after being activated by target antigens.

In some embodiments, the present invention also provides a nucleic acid encoding the chimeric T cell receptor of any of the foregoing or the first polypeptide and the second polypeptide.

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

(1) The variable region of the antibody heavy chain, the extracellular segment, the transmembrane region and the intracellular end of the constant region of the T cell receptor (TCR) α chain, the linker, the variable region of the antibody light chain, and the T cell Receptor (TCR) beta chain constant region extracellular segment, transmembrane region and intracellular terminus; or,

(2) The variable region of the antibody heavy chain, the extracellular segment, the transmembrane region and the intracellular end of the constant region of the T cell receptor (TCR) beta chain, the linker, the variable region of the antibody light chain, and the T cell Receptor (TCR) alpha chain constant region extracellular segment, transmembrane region and intracellular terminus; or,

(3) The variable region of the antibody light chain, the extracellular segment, the transmembrane region and the intracellular end of the constant region of the T cell receptor (TCR) α chain, the linker, the variable region of the antibody heavy chain, and the T cell Receptor (TCR) beta chain constant region extracellular segment, transmembrane region and intracellular terminus; or,

(4) The variable region of the antibody light chain, the extracellular segment, the transmembrane region and the intracellular end of the constant region of the T cell receptor (TCR) beta chain, the linker, the variable region of the antibody heavy chain, and the T cell Receptor (TCR) alpha chain constant region extracellular segment, transmembrane region and intracellular terminus; or,

(5) Replace the T cell receptor (TCR) α chain in (1)-(4) with the T cell receptor (TCR) γ chain, and the T cell receptor (TCR) β chain with the T cell receptor ( TCR) delta chain corresponding nucleic acid.

In some specific embodiments, the T cell receptor (TCR) alpha or beta chain constant region is derived from a human TCR alpha or beta constant region, and may also be derived from a murine TCR alpha or beta constant region.

In some embodiments, the alpha chain and/or beta chain of the TCR can be modified with amino acid point mutations, polypeptide fragment substitution modifications to reduce mismatch with endogenously expressed T cell receptors. Preferably, the alpha chain and/or beta chain of the TCR is subjected to cysteine point mutation. Specifically, the 48th amino acid in the constant region of the TCRα chain is mutated to cysteine, and the 57th amino acid in the constant region of the TCRβ chain is mutated to cysteine, so that there is an additional link between the first peptide chain and the second peptide chain. connected by disulfide bonds.

More preferably, the nucleotide sequence of the human TCRα chain constant region cysteine mutant is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 11; the human TCRβ chain constant region cysteine mutant nucleoside The acid sequence is SEQ ID NO:2, and the amino acid sequence is SEQ ID NO:12.

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

Further, there are four forms of the first amino acid in the constant region of the natural human TCRα chain; the first amino acid is Asp, that is, the polypeptide shown in SEQ ID NO: 11, and the first amino acid is the polypeptide of Asn, His or Tyr, so the human TCRα chain The constant region cysteine mutant can also be selected from the polypeptides represented by the following sequences:

①The amino acid sequence is SEQ ID NO:31, and the corresponding nucleotide sequence is SEQ ID NO:25;

②The amino acid sequence is SEQ ID NO:32, and the corresponding nucleotide sequence is SEQ ID NO:26;

③ The amino acid sequence is SEQ ID NO:33, and the corresponding nucleotide sequence is SEQ ID NO:27.

More preferably, the nucleotide sequence of the murine TCRα chain constant region cysteine mutant is SEQ ID NO:7, and the amino acid sequence is SEQ ID NO:17; the murine TCRβ constant region cysteine mutant nucleotide is The sequence is SEQ ID NO:8, and the amino acid sequence is SEQ ID NO:18.

Further, there are four forms of the first amino acid in the constant region of the natural mouse-derived TCRα chain; the first amino acid is Asn, that is, the polypeptide shown in SEQ ID NO: 17, and the first amino acid is the polypeptide of Asp, His or Tyr, so the mouse-derived TCRα chain The constant region cysteine mutant can also be selected from the polypeptides represented by the following sequences:

①The amino acid sequence is SEQ ID NO:34, and the corresponding nucleotide sequence is SEQ ID NO:28;

②The amino acid sequence is SEQ ID NO:35, and the corresponding nucleotide sequence is SEQ ID NO:29;

③ The amino acid sequence is SEQ ID NO:36, and the corresponding nucleotide sequence is SEQ ID NO:30.

In some embodiments, the T cell receptor (TCR) alpha chain can be replaced by a T cell receptor (TCR) gamma chain; the T cell receptor (TCR) beta chain can be replaced by a T cell receptor (TCR) delta chain. Preferably, the T cell receptor (TCR) γ chain and the T cell receptor (TCR) δ chain can be derived from human TCR γ chain or δ chain constant region, and can also be derived from murine TCR γ chain or δ chain constant region.

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

In some embodiments, according to any one of the aforementioned chimeric T cell receptors, there is provided an effector cell expressing any one of the aforementioned chimeric T cell receptors or the aforementioned complexes on its cell surface. In some embodiments, the effector cell comprises a nucleic acid encoding the chimeric T cell receptor. In some embodiments, the T cells are 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 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 of the above-described embodiments and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising an effector cell expressing any one of the chimeric T cell receptors described above and a pharmaceutically acceptable carrier.

The chimeric T cell receptor of the present invention and the chimeric T cell receptor-transfected cells of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The chimeric T cell receptors, chimeric T cell receptor complexes 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, usually in a sealed container, as part of a kit. Such kits, but not necessarily, include instructions for use. It may comprise a plurality of such unit dosage forms.

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

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MIR (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon administration. These vectors are well known to those of ordinary skill in the art. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof. Pharmaceutically acceptable carriers in therapeutic compositions can contain 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, therapeutic compositions can be prepared as injectables, eg, liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid carriers, prior to injection can also be prepared.

Once formulated, the compositions of the present invention can 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, especially a human.

When the pharmaceutical composition of the present invention is used for actual treatment, various pharmaceutical compositions in different dosage forms can be adopted according to the usage. Preferably, injections, oral preparations and the like can be exemplified. These pharmaceutical compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.

The pharmaceutical compositions of the present invention can also be administered in the form of sustained release formulations. For example, the polypeptides of the present invention can be incorporated into pellets or microcapsules with a sustained release polymer as a carrier, and the pellets or microcapsules are then surgically implanted into the tissue to be treated.

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

The chimeric T cell receptors of the present invention can be used as pharmaceuticals or diagnostic reagents. Modifications or other improvements can be made to obtain characteristics that are more suitable for use as pharmaceuticals or diagnostic reagents.

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

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

Methods of manufacture, articles of manufacture, and kits suitable for the methods described herein are also provided.

In some embodiments, there is also provided the use of a chimeric T cell receptor of any one of the above-described chimeric T cell receptors in the manufacture of a kit for treating or diagnosing a target antigen-related 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 combining the target cell with a chimeric T cell receptor expressing any one of the above-described chimeric T cell receptors Effector cell contacts 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, according to any of the above-described methods of target cell killing, the contacting is in vivo. In some embodiments, the contacting is in vitro.

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

In some embodiments, there is provided a method of treating a target antigen-related disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising effector T cells comprising effector T cells that specifically bind to a target A chimeric T cell receptor for an antigen, comprising: a) a first polypeptide fused to a variable region of an antibody heavy chain; b) a second polypeptide fused to a variable region of an antibody light chain; wherein the antibody heavy chain can be The 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 aforementioned chimeric T cell receptors.

In some embodiments, there is provided a method of treating a T cell-mediated disorder that occurs due to cell, tissue, body part or organ transplantation in a subject in need thereof, comprising the step of: transferring the cell, tissue, body part Or an organ is transplanted into the subject; and two or more doses of a pharmaceutically acceptable amount of any one of the foregoing chimeric T cell receptors are administered to the subject.

In some embodiments, according to any of the above methods, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma , head and neck cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer , sarcoma, gastric cancer, uterine cancer and thyroid cancer. In some embodiments, the target antigen-related 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 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 immune Deficiency 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, Kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer , uterine cancer and thyroid cancer.

In some embodiments, there is provided a method of treating a target antigen-related 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 encoding a chimeric T cell receptor as described above A nucleic acid of any one of the chimeric T cell receptors.

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

In some embodiments, the present invention provides any of the aforementioned chimeric T cell receptors, the complexes, nucleic acids, vectors or effector cells in the manufacture of kits, preparations or a target antigen-related disease for the treatment or diagnosis of an individual in need thereof. Use in pharmaceutical compositions.

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

Specifically, the cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, Head and neck cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, Sarcoma, gastric cancer, uterine cancer, and thyroid cancer; 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).

The classic CAR molecule consists 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 T cells. Among them, the single-chain antibody region includes the heavy chain variable region and the light chain variable region of the antibody (in some cases, it also includes the IgG CH1 region, which plays a structural role), and the two are connected by a flexible linking peptide. The intracellular signal region is composed of co-stimulatory signal molecules (4-1BB, CD28, etc.) and the signal molecule CD3ζ in series.

After the chimeric T cell receptor constructed in the present application is expressed in cells, two polypeptide chains are covalently linked together via disulfide bonds, wherein the first polypeptide is composed of the variable region ( _VH ) of the heavy chain of the antibody. It is formed by fusion with TCRα constant region (Cα), and the second polypeptide is formed by fusion of antibody light chain variable region ( _VL ) and _TCRβ constant region (Cβ); chimeric T cell receptor can interact with T cells The source expressed CD3 subunits (ε, δ, λ, ζ) form a complex to function. The gene sequence of the chimeric T cell receptor is connected by the furin and p2A protease cleavage site polypeptide segments. The two polypeptide chains will be transcribed and translated into proteins together, and then cleaved into two independent two by the protease corresponding to furin and p2A. protein. Chimeric T cell receptors are available in various combinations: antibody heavy chain variable region ( _VH ) fused to TCRα constant region (Cα), antibody light chain variable region ( _VL ) fused to _TCRβ constant region (Cβ) , or the antibody light chain variable region ( _VL ) is fused to the TCRα constant region (Cα), the antibody heavy chain variable region ( _VH ) is fused to the _TCRβ constant region (Cβ); and the two exchange relative furin and P2A sequence before and after. The variable regions of light and heavy chains of antibodies can be replaced with variable regions of antibodies of various specificities, such as anti-EGFR, CD19, CD20, and the like. Various variants of the TCRα and β constant regions are also available, including wild-type TCRα and β constant regions, cysteine single point mutant TCRα and β constant regions, human-mouse chimeric TCRα and β constant regions, and half-containing TCRα and β constant regions. Cystine single point mutated human-mouse chimeric TCR alpha and beta constant regions.

The present invention obtains a highly stable and highly specific T cell receptor, which can be used for disease diagnosis and treatment. During the TCR design process, the inventors creatively employed multiple strategies. The present application is modified by introducing disulfide bonds in the constant region of the TCR molecule. In theory, there are many sites that can form artificial interchain disulfide bonds in TCR, but finding suitable sites to form artificial interchain disulfide bonds in TCR enables TCRs containing artificial interchain disulfide bonds to be successfully complexed. It is very difficult to obtain high-yield, highly stable TCRs with specific binding activity to their original ligands. Those skilled in the art are committed to developing TCRs containing artificial interchain disulfide bonds that can be well renatured, refolded, purified, have high stability, high renaturation yield and can specifically bind to their original ligands . The invention can reduce the new reactivity of TCR-T cells and reduce the mismatch by carrying out the modification of the disulfide bond at the specific site.

Although CAR-T therapy has been reported frequently, the safety issue is the focus of the industry and the FDA. Avoiding self-activation and reducing side effects is an urgent problem to be solved. The present invention introduces the antibody antigen-binding fragment and the original TCR for fusion to form a chimeric T cell receptor (STAR) combined with the antibody-T cell receptor, further improves the pairing of the α chain and the β chain, and improves the binding of the TCR. More importantly, the transformation of the original TCR in vivo is less than that of CAR-T, which reduces the introduction of exogenous amino acids, reduces the risk of side effects, and improves safety. After the verification of chimeric T cell receptors with multiple targets, the results show that chimeric T cell receptors can mediate the activation of T cells after antigen stimulation, and are compatible with the corresponding antibody-chimeric antigen receptor (CAR) obtained. ), the degree of antigen activation is similar, and more importantly, in the resting state without antigen stimulation, the chimeric T cell receptor has no self-activation phenomenon, while CAR has a high self-activation phenomenon.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here. The present invention will be described in further detail below in conjunction with the examples.

Description of drawings

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

Figure 1 Schematic diagram of the structure of STAR and CAR molecules. After STAR is expressed in cells, two polypeptide chains are covalently linked together by disulfide bonds, wherein the first polypeptide chain is fused by the antibody heavy chain variable region (VH) and the TCRα constant region (Cα), The second polypeptide chain is composed of antibody light chain variable region (VL) and TCRβ constant region (Cβ) fusion; STAR can form a complex with CD3 subunits (ε, δ, λ, ζ) endogenously expressed in T cells body functions.

Figure 2 Schematic diagram of the molecular gene structure sequence of STAR and CAR. The STAR gene sequence is connected by the furin and P2A protease cleavage site polypeptide segments, the two polypeptide chains will be transcribed, translated and expressed into proteins, and then cleaved into two independent proteins by the protease corresponding to furin and p2A. STARs are available in various combinations: antibody heavy chain variable region (VH) fused to TCRα constant region (Cα), antibody light chain variable region (VL) fused to TCRβ constant region (Cβ), or antibody light chain variable region (VL) is fused to the TCRα constant region (Cα), the antibody heavy chain variable region (VH) is fused to the TCRβ constant region (Cβ); and the two exchange the relative order of furin and P2A.

Figure 3. Epithelial profile of EGFR-targeting STAR in human T cells. The STAR gene was introduced into the human T cell line Jurkat Clone 5 (endogenous TCR-deleted Jurkat subclone) using a lentiviral vector. Cells 3 days after infection were taken, stained with anti-human TCR-α/β flow antibody, and then subjected to flow detection. It can be found that STAR can be stained by anti-TCR-α/β antibody, and the staining level is comparable to that of native E1-TCR, compared with non-transgenic negative control cells. This result suggests that STAR molecules can be membrane-bound and that their alpha and beta chains can pair.

Figure 4. Ability of EGFR-targeting STARs to bind antigen when on the surface of human T cell membranes. The Jurkat Clone 5 cells 3 days after the above-mentioned STAR gene was introduced were stained with the antigenic proteins EGFR-His and anti-His-APC by flow cytometry, and then flow cytometry was performed. It was found that STAR showed stronger staining compared to the native E1-TCR (not specific for EGFR) negative control cells, and the staining level was comparable to the anti-EGFR CAR.

Figure 5. Ability of EGFR-targeting STARs to mediate T cell activation. JurkatClone 5 cells 3 days after the above-mentioned STAR gene introduction were cultured in EGFR antigen-coated cell culture plates and co-incubated with tumor cells A549 (EGFR-positive human lung cancer cell line). After 24 hours, cells were harvested and stained with anti-human CD69-FITC antibody for flow cytometry. The CD69-positive cells on the abscissa are cells that express the T cell activation marker CD69 molecule. It can be found that STAR can make T cells express the activation marker of CD69 under antigen stimulation, that is, STAR can mediate the activation of T cells after antigen stimulation, and the activation degree is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.

Figure 6. Function of EGFR-targeting STARs in human primary T cells. Human peripheral blood cells were obtained, and CD4+ and CD8+ T cells were purified using pan T cell isolation kit. Then the T cells were stimulated and activated with anti-CD3/CD28 antibody for 72 hours, and then the STAR gene was transferred into T cells with a lentiviral vector. After virus infection, culture to sufficient numbers in RPMI 1640 medium containing 20% serum and 200 IU/mL IL-2. T cells were co-cultured with A431 cells, an EGFR-positive human skin cancer cell, to detect T cell activation and target cell death. After 8 hours of co-culture, T cells were taken for staining, and it was found that both STAR and CAR could mediate T cell activation. STAR induced significant T cell activation as seen from the levels of the T cell marker CD69 (Fig. 6a) and the T cell cytokine IFN-γ (Fig. 6b). After 24 hours of co-culture, the supernatant of cells was taken to detect the level of lactate dehydrogenase (LDH) (Fig. 6c), which could reflect the death of target cells. The results showed that both STAR-T and CAR-T cells had obvious killing effects on target cells.

Figure 7 Ability of STARs 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 after 3 days of infection were collected and incubated with tumor cells Raji, Mino and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were harvested and stained with anti-humanCD69-FITC flow cytometry antibody, followed by flow cytometry. The CD69-positive cells on the ordinate are cells expressing the T cell activation marker CD69 molecule. It can be found that STAR can make T cells express the activation marker of CD69 under antigen stimulation, that is, STAR can mediate the activation of T cells after antigen stimulation, and the activation degree is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while 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 obtained, and CD4+ and CD8+ T cells were purified using pan T cell isolation kit. T cells were then stimulated and activated with anti-CD3/CD28 antibody for 72 hours, and then the STAR gene targeting CD19 was transferred into T cells with a lentiviral vector. After virus infection, culture to sufficient numbers in RPMI 1640 medium containing 20% serum and 200 IU/mL IL-2. T cells were co-cultured with Raji and LY-1 cells to detect target cell death. After 8 hours of co-culture, T cells were taken for staining, and it was found that both STAR and CAR could mediate T cell activation. From the level of the T cell cytokine IFN-γ (Fig. 4), STAR induced significant T cell activation (higher than that of CAR). The results of the expression level of IFN-γ in T cells showed that the target cells had a significant activation effect on STAR-T cells.

Figure 9 Ability of STARs 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 after 3 days of infection were collected and incubated with tumor cells Raji, Mino and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were harvested and stained with anti-humanCD69-FITC flow cytometry antibody, followed by flow cytometry. The CD69-positive cells on the ordinate are cells expressing the T cell activation marker CD69 molecule. It can be found that STAR can make T cells express the activation marker of CD69 under antigen stimulation, that is, STAR can mediate the activation of T cells after antigen stimulation, and the activation degree is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while 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 obtained, and CD4+ and CD8+ T cells were purified using pan T cell isolation kit. T cells were then stimulated and activated with anti-CD3/CD28 antibody for 72 hours, and then the STAR gene targeting CD20 was transferred into T cells with a lentiviral vector. After virus infection, culture to sufficient numbers in RPMI 1640 medium containing 20% serum and 200 IU/mL IL-2. T cells were co-cultured with Raji and LY-1 cells to detect target cell death. After 8 hours of co-culture, T cells were taken for staining, and it was found that both STAR and CAR could mediate T cell activation. From the level of the T cell cytokine IFN-γ (Fig. 4), STAR induced significant T cell activation (higher than that of CAR). The results of the expression level of IFN-γ in T cells showed that the target cells had a significant activation effect on STAR-T cells.

Detailed ways

Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology, which are within the purview of those skilled in the art.

Embodiments of the present invention are described in detail below in order to provide those of ordinary skill in the art with a full disclosure and description of how to conduct and utilize the testing, screening, and therapeutic methods of the present invention. It should be noted that these embodiments are only illustrative, It should not be construed as a limitation of the present invention.

Example 1. Construction of EGFR-targeted STAR

The specific construction method is as follows:

1. TCR constant region sequence determination

The constant regions (C regions) of the α chain and β chain of TCR in STAR are derived from the cDNA of human peripheral blood T cells and cloned by PCR molecules; The 48th and 57th amino acids of the constant region were mutated to cysteine to help form an additional disulfide bond between the α chain and the β chain, increasing their mutual pairing efficiency, and named E1-TCR.

2. Sequence determination of antibodies targeting EGFR

The variable region of antibody heavy chain (VH) and the variable region of antibody light chain (VL) are Cetuximab (Cetuximab, Cetux for short), which is only used as an example to explain the content of the present invention, and other known antibodies can be substituted.

3. Construction of STAR targeting EGFR

STAR contains two polypeptide chains, the first polypeptide chain is fused with Cetux-VL and TCRβ chain, and the second polypeptide chain is fused with Cetux-VH and α chain. The gene sequence of STAR is connected by the furin and p2A protease cleavage site polypeptide segments. The two polypeptide chains will be transcribed and translated into a fusion polypeptide, and then cleaved by the protease corresponding to furin and p2A into two independent protein subunits. The two subunits are covalently bound by disulfide bonds and form complexes with the endogenous CD3 subunits (ε, δ, γ, ζ) of T cells (as shown in Figures 1 and 2).

The entire gene was inserted into the lentiviral expression vector pHAGE through the restriction endonuclease sites NheI and NotI. The vector carries ampicillin resistance, EF1α promoter and IRES-RFP fluorescent reporter gene.

4. Cloning and assembly of gene fragments

The obtained four fragments "Cetux VL", "TCRβ-C", "Cetux-VH", "TCRα-C" were cloned from pHAGE-Cetux-28zCAR vector and pHAGE-E1-TCR vector, respectively. Each pair of primers has a 25bp base homologous to the front and back, and the four fragments are connected to the lentiviral vector by one-step recombination by the Gibson Assembly method. to get a STAR.

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

The nucleotide sequence of the E1 TCRβ 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 Gibson Assembly was transformed into the DH5α strain and allowed to grow overnight on LB plates containing ampicillin. Single clones were picked for sequencing, and the sequencing primers were seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier.

6. Plasmid Extraction

The bacteria with correct sequencing results were inoculated in LB liquid medium and cultured overnight. Extract the plasmid using an endotoxin-removing kit. The plasmid concentration was measured by Nanodrop, the final plasmid concentration was around 1000ng/ul, and the A260/A280 value was greater than 1.8.

Example 2. Functional verification of STAR targeting EGFR

1. Lentiviral Packaging

The pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were proportionally transfected into 293T cells (transfection using PEI). Collect the cell culture supernatant from 48 hours and 72 hours, mix the supernatant with PEG8000, let it stand overnight and then centrifuge to obtain the virus pellet. Resuspend with a small volume of medium to concentrate the virus.

2. Lentiviral Infection of Human T Cell Lines

Jurkat clone 5 cells (Jurkat subclones with endogenous TCR deletion) were infected with the lentivirus carrying the target gene. The concentrated lentivirus and the transfer agent Polybrene were added to the T cell culture medium, and the infection was centrifuged at 1500 rpm at 32°C for 2 hours. Three days after infection, fluorescent reporter genes can be observed and target protein expression can be detected.

3. Detection of the upper membrane and antigen binding capacity of STAR targeting EGFR

T cells after 3 days of infection were taken and stained with anti-human TCRα/β-BV421 flow antibody, and then flow cytometry was performed. It can be found (Fig. 3) that STAR can be stained by anti-TCRα/β antibody compared to non-transgenic negative control cells, and the staining level is comparable to that of native E1-TCR. This result suggests that STAR molecules can be membrane-bound and that their alpha and beta chains can pair.

The T cells after 3 days of infection were taken and stained with the antigenic proteins EGFR-His and anti-His-APC by flow cytometry, and then flow cytometry was performed. It can be found (Fig. 4) that STAR showed stronger staining compared to the negative control cells of the native E1-TCR (specific not to EGFR), and the staining level was comparable to that of the anti-EGFR CAR. This result shows that STAR has antigen recognition and binding ability comparable to CAR molecules.

4. Co-incubation of EGFR-targeted STAR-T cells with target cells and detection of their ability to mediate T cell activation

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). After 24 hours, cells were harvested and stained with anti-human CD69-FITC flow cytometry antibody, followed by flow cytometry (Figure 5). The CD69-positive cells on the abscissa are cells that express the T cell activation marker CD69 molecule. It can be found that STAR can make T cells express the activation marker of CD69 under antigen stimulation, that is, STAR can mediate the activation of T cells after antigen stimulation, and the activation degree is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.

5. Isolation, Culture and Lentiviral Infection of Human Primary T Cells

Human peripheral blood cells were obtained, and CD4 and CD8 T cells were purified by using the whole T cell magnetic bead separation kit. T cells were then stimulated and activated in culture dishes coated with anti-CD3/CD28 antibodies for 48-72 hours, and T cells were observed to increase in size, grow in aggregates, and polarize their shapes. At this time, the target gene was transferred into T cells with a lentiviral vector, and the infection method was centrifugation at 1500 rpm at 32°C for 2 hours. After virus infection, culture to sufficient numbers in RPMI 1640 medium containing 20% serum and 200 IU IL-2.

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

T cells were co-cultured with A431 cells (a highly EGFR-positive human skin cancer cell) at a ratio of 1:1 to 5:1 to detect T cell activation and target cell death. After 8 hours of co-culture, T cells were taken for staining, and it was found that both STAR and CAR could mediate T cell activation. STAR induced significant T cell activation as seen from the levels of the T cell marker CD69 (Fig. 6a) and the T cell cytokine IFN-γ (Fig. 6b). After 24 hours of co-culture, the supernatant of cells was taken to detect the level of lactate dehydrogenase (LDH) (Fig. 6c), which could reflect the death of target cells. The results showed that STAR-T cells had obvious killing effect on target cells.

Example 3. Construction of STAR targeting CD19

The specific construction method is as follows:

1. TCR constant region sequence determination

The cDNAs derived from human peripheral blood T cells or mouse spleen T cells were cloned by PCR molecules; on the basis of the original TCR sequence, the 48th and 57th amino acid sites of the constant regions of the α chain and β chain were mutated respectively. It is cysteine to help form an additional disulfide bond between the α chain and the β chain, increasing their mutual pairing efficiency, and named as E1-TCR (human source) or E11-TCR (mouse source).

2. Determination of the sequence of antibodies targeting CD19

The variable region of antibody heavy chain (VH) and the variable region of antibody light chain (VL) were selected as scFv fragments of CD19-specific mouse monoclonal antibody (clone No. FMC63), only as an example to explain the content of the present invention, other known antibodies are Can be replaced.

3. Construction of STAR targeting CD19

STAR contains two polypeptide chains. FMC63-VL and TCRβ chain are fused as the first polypeptide chain, and FMC63-VH and α chain are fused as the second polypeptide chain. The gene sequence of STAR is connected by the furin and p2A protease cleavage site polypeptide segments. The two polypeptide chains will be transcribed and translated into a fusion polypeptide, and then cleaved by the protease corresponding to furin and p2A into two independent protein subunits. The two subunits are covalently bound by disulfide bonds and form a complex with the endogenous CD3 subunits (ε, δ, γ, ζ) of T cells.

The entire gene was inserted into the lentiviral expression vector pHAGE through the restriction endonuclease sites NheI and NotI. The vector carries ampicillin resistance, EF1α promoter and IRES-RFP fluorescent reporter gene.

4. Cloning and assembly of gene fragments

The obtained four fragments "FMC63-VL", "TCRβ-C", "FMC63-VH", "TCRα-C" were cloned from pHAGE-FMC63-41BBzCAR vector and pHAGE-E1-TCR vector, respectively. Each pair of primers has a 25bp base homologous to the front and back, and the four fragments are connected to the lentiviral vector by one-step recombination by the Gibson Assembly method. to get a STAR.

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

The nucleotide sequence of the E1 TCRβ chain constant region cysteine mutant is SEQ ID NO: 2;

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

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

5. Vector Transformation and Sequencing

The product of Gibson Assembly was transformed into the DH5α strain and allowed to grow overnight on LB plates containing ampicillin. Single clones were picked for sequencing, and the sequencing primers were seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier.

6. Plasmid Extraction

The bacteria with correct sequencing results were inoculated in LB liquid medium and cultured overnight. Extract the plasmid using an endotoxin-removing kit. The plasmid concentration was measured by Nanodrop, the final plasmid concentration was around 1000ng/ul, and the A260/A280 value was greater than 1.8.

Example 4. Functional verification of STAR targeting CD19

1. Lentiviral Packaging

The pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were proportionally transfected into 293T cells (transfection using PEI). Collect the cell culture supernatant of 48 hours and 72 hours, mix the supernatant with PEG8000, let it stand overnight and then centrifuge to obtain the virus pellet. Resuspend with a small volume of medium to concentrate the virus.

2. Lentiviral Infection of Human T Cell Lines

Jurkat clone 5 cells (Jurkat subclones with endogenous TCR deletion) were infected with the lentivirus carrying the target gene. The concentrated lentivirus and the transfer agent Polybrene were added to the T cell culture medium, and the infection was centrifuged at 1500 rpm at 32°C for 2 hours. Three days after infection, fluorescent reporter genes can be observed and target protein expression can be detected.

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

T cells after 3 days of infection were collected and incubated with tumor cells Raji, Mino and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were harvested and stained with a flow antibody against anti-human CD69-FITC, followed by flow detection (Figure 7). The CD69-positive cells on the ordinate are cells expressing the T cell activation marker CD69 molecule. It can be found that STAR can make T cells express the activation marker of CD69 under antigen stimulation, that is, STAR can mediate the activation of T cells after antigen stimulation, and the activation degree is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.

4. Isolation, Culture and Lentiviral Infection of Human Primary T Cells

Obtain human peripheral blood cells, and purify CD3 ⁺ T cells from them using the Whole T Cell Magnetic Bead Isolation Kit. T cells were then stimulated and activated in culture dishes coated with anti-CD3/CD28 antibodies for 48-72 hours, and T cells were observed to increase in size, grow in aggregates, and polarize their shapes. At this time, the target gene was transferred into T cells with a lentiviral vector, and the infection method was centrifugation at 1500 rpm at 32°C for 2 hours. After virus infection, culture to sufficient numbers in RPMI 1640 medium containing 20% serum and 200 IU/mL IL-2.

5. Functional validation of STAR targeting CD19 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 to detect T cell activation and target cell death. After 8 hours of co-culture, T cells were taken for staining, and it was found that both STAR and CAR could mediate T cell activation. STAR induced significant T cell activation as seen from the levels of the T cell cytokine IFN-[gamma] (Fig. 8). The results of the expression level of IFN-γ in T cells showed that the target cells had a significant activation effect on STAR-T cells.

Example 5. Construction of STAR targeting CD20

The specific construction method is as follows:

1. Determination of the sequence of antibodies targeting CD20

The variable region of the antibody heavy chain (VH) and the variable region of the antibody light chain (VL) were selected as scFv fragments of the CD20-specific antibody Ofatumumab (ofatumumab, OFA), only as an example to explain the content of the present invention, other known Antibodies can be substituted.

2. Construction of STAR targeting CD20

STAR contains two polypeptide chains. OFA-VL and TCRβ chain are fused as the first polypeptide segment, and OFA-VH and α chain are fused as the second polypeptide segment. The gene sequence of STAR is connected by the furin and p2A protease cleavage site polypeptide segments. The two polypeptide chains will be transcribed and translated into a fusion polypeptide, and then cleaved by the protease corresponding to furin and p2A into two independent protein subunits. The two subunits are covalently bound by disulfide bonds and form a complex with the endogenous CD3 subunits (ε, δ, γ, ζ) of T cells.

The entire gene was inserted into the lentiviral expression vector pHAGE through the restriction endonuclease sites NheI and NotI. The vector carries ampicillin resistance, EF1α promoter and IRES-RFP fluorescent reporter gene.

3. Cloning and assembly of gene fragments

The obtained four fragments "OFA-VL", "TCRβ-C", "OFA-VH", "TCRα-C" were cloned from pHAGE-OFA-41BBzCAR vector and pHAGE-E11-TCR vector, respectively. Each pair of primers has a 25bp base homologous to the front and back, and the four fragments are connected to the lentiviral vector by one-step recombination by the Gibson Assembly method. to get a STAR.

The nucleotide sequence of the E11 TCRα chain constant region cysteine mutant is SEQ ID NO: 7;

The nucleotide sequence of the E11 TCRβ 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

The product of Gibson Assembly was transformed into the DH5α strain and allowed to grow overnight on LB plates containing ampicillin. Single clones were picked for sequencing, and the sequencing primers were seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier.

5. Plasmid Extraction

The bacteria with correct sequencing results were inoculated in LB liquid medium and cultured overnight. Extract the plasmid using an endotoxin-removing kit. The plasmid concentration was measured by Nanodrop, the final plasmid concentration was around 1000ng/ul, and the A260/A280 value was greater than 1.8.

Example 6. Functional verification of STAR targeting CD20

1. Lentiviral Packaging

The pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were proportionally transfected into 293T cells (transfection using PEI). Collect the cell culture supernatant of 48 hours and 72 hours, mix the supernatant with PEG8000, let it stand overnight and then centrifuge to obtain the virus pellet. Resuspend with a small volume of medium to concentrate the virus.

2. Lentiviral Infection of Human T Cell Lines

Jurkat clone 5 cells (Jurkat subclones with endogenous TCR deletion) were infected with the lentivirus carrying the target gene. The concentrated lentivirus and the transfer agent Polybrene were added to the T cell culture medium, and the infection was centrifuged at 1500 rpm at 32°C for 2 hours. Three days after infection, fluorescent reporter genes can be observed and target protein expression can be detected.

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

T cells after 3 days of infection were collected and incubated with tumor cells Raji, Mino and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were harvested and stained with anti-human CD69-FITC flow cytometry antibody, followed by flow cytometry (Figure 9). The CD69-positive cells on the ordinate are cells expressing the T cell activation marker CD69 molecule. It can be found that STAR can make T cells express the activation marker of CD69 under antigen stimulation, that is, STAR can mediate the activation of T cells after antigen stimulation, and the activation degree is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.

4. Isolation, Culture and Lentiviral Infection of Human Primary T Cells

Obtain human peripheral blood cells, and purify CD3 ⁺ T cells from them using the Whole T Cell Magnetic Bead Isolation Kit. T cells were then stimulated and activated in culture dishes coated with anti-CD3/CD28 antibodies for 48-72 hours, and T cells were observed to increase in size, grow in aggregates, and polarize their shapes. At this time, the target gene was transferred into T cells with a lentiviral vector, and the infection method was centrifugation at 1500 rpm at 32°C for 2 hours. After virus infection, culture to sufficient numbers in RPMI 1640 medium containing 20% serum and 200 IU/mL IL-2.

5. Functional validation of STAR targeting CD20 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 to detect T cell activation and target cell death. After 8 hours of co-culture, T cells were taken for staining, and it was found that both STAR and CAR could mediate T cell activation. STAR induced significant T cell activation as seen from the levels of the T cell cytokine IFN-[gamma] (Figure 10). The results of the expression level of IFN-γ in T cells showed that the target cells had a significant activation effect on STAR-T cells.

It can be seen that the present invention has successfully constructed STAR with multiple targets, and successfully verified that the antibody-T cell chimeric receptor involved in this application can interact with the endogenously expressed CD3 subunits (ε, δ, λ) of T cells. , ζ) to form a complex to function, which can mediate the activation of T cells after antigen stimulation, and compared with the corresponding antibody-chimeric antigen receptor (CAR) obtained by preparation, the degree of antigen activation is similar, and more importantly, In the resting state without antigenic stimulation, STAR has no self-activation phenomenon, while CAR has a high self-activation phenomenon. Due to space limitations, the present invention lists exemplary STARs, which are sufficient to support the successful construction and outstanding technical effects of the STARs of the present invention.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms 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.

Although embodiments of the present 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 in these embodiments without departing from the principles and spirit of the invention, The scope of the invention is defined by the claims and their equivalents.

Claims (25)

1. 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.

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:

(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 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,

(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.

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).

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