CN110818802B

CN110818802B - Chimeric T cell receptor STAR and application thereof

Info

Publication number: CN110818802B
Application number: CN201810898720.2A
Authority: CN
Inventors: 刘玥; 王嘉盛; 刘光娜; 赵学强; 林欣
Original assignee: Shanghai Icell Biotechnology Co ltd; Tsinghua University; Bristar Immunotech Ltd
Current assignee: Shanghai Icell Biotechnology Co ltd; Tsinghua University; Bristar Immunotech Ltd
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2022-02-08
Anticipated expiration: 2038-08-08
Also published as: CN110818802A; 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 formed by two different peptide chains and consists of alpha and beta peptide chains, each peptide chain can be divided into a variable region (V region) and a constant region (C region), and the constant region comprises an extracellular region, a transmembrane region and an intracellular terminal; it is characterized by a short intracellular region. TCR molecules belong to the immunoglobulin superfamily, with antigen-specific presence in the V region. TCRs are divided into two categories: TCR1 and TCR 2; TCR1 consists of two chains, γ and δ, and TCR2 consists of two chains, α and β. In peripheral blood, 90% to 95% of T cells express TCR 2; furthermore, either T cell expressed only 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 existing TCR-T therapy, endogenous TCR is generally required to be separated and engineered, and is introduced into brand new T cells and infused back into a human body, so that the number of the T cells with the capability of targeting cancer cells is greatly increased, and the TCR-T therapy is expected to identify and attack various solid tumors and blood tumors. However, exogenous TCR α and β chains and endogenous α and β chains will recognize each other and pair to form hybrid TCRs. This TCR mismatch phenomenon is thought to be widely present in TCR-T therapy and may cause a decrease in TCR expression on the cell surface and a decrease in cell activity. Mismatched TCRs may also form a new TCR with unknown specificity that can be associated with autoimmune (on-target) or cross-reactive (off-target) toxicity in the TCR-T. Although there is no clinical evidence of autoreactive disease caused by TCR mismatch at present, the autoimmune risk posed by heterozygous TCRs is not negligible. Therefore, the expression and paired assembly of TCR in T cells and affinity for pMHC are both key factors affecting the full anti-tumor capacity of TCR gene therapy.

Therefore, how to modify the TCR gene to enable the TCR α chain and the TCR β chain to be correctly paired on the surface of a T cell, and the expression efficiency and affinity thereof are both enhanced, and meanwhile, avoiding side effects and improving safety become one of the hot spots of TCR gene therapy in recent years. The main strategies include increasing the affinity of the TCR in T cells, optimizing the pairing of TCR chains, and enhancing its surface expression efficiency. For example, the following methods were used by researchers to reduce TCR mismatches: introducing a disulfide bond in the TCR chain; replacing the C region of the human TCR molecule with a murine conserved C region; inverting amino acid residues of conserved structural domains at the interfaces of the TCR alpha chain and the beta chain; constructing a single chain TCR (scTCR) chimera; fusing the C region of the TCR chain to the CD3 molecule; the a beta TCR gene is transferred into gamma delta T cells, but the effect is not satisfactory. It can be seen that although modifications to TCR-T to optimize the pairing strategy of exogenous TCR molecules have been proposed, there are still many places where improvements are needed.

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, according to the foregoing 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 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 gamma 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 may be subjected to any amino acid sequence modification, including but not limited to amino acid point mutation modification, polypeptide fragment substitution modification, to reduce mismatches with an endogenously expressed T cell receptor. For example: the 48 th amino acid of the constant region of the TCR alpha chain is mutated into cysteine, and the 57 th amino acid of the constant region of the TCR alpha chain is mutated into cysteine, so that the first polypeptide and the second polypeptide are connected through a disulfide bond; for another example: the 85 th amino acid of the constant region of the TCR alpha chain is mutated into alanine, and the 88 th amino acid of the constant region of the TCR alpha chain is mutated into glycine;

specifically, (1) the first subunit of the T cell receptor is a TCR alpha 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 beta 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 alpha 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 beta 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 alpha chain or beta chain, respectively, to obtain a VH-TCR alpha chain fusion or a VL-TCR beta chain fusion.

Further preferably, the VH and VL derived from IMCC225 (Cetuximab, Cetuximab/Cetux), rituximab (rituximab), Ofatumumab (Ofatumumab), CD19 monoclonal antibody FMC63 are fused to the TCR α chain constant region or 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) the antibody heavy chain variable region, the T Cell Receptor (TCR) alpha chain constant region extracellular section, the transmembrane region and the intracellular tail end, the linker, the antibody light chain variable region, the T Cell Receptor (TCR) beta chain constant region extracellular section, the transmembrane region and the intracellular tail end are sequentially arranged; or the like, or, alternatively,

(2) the antibody heavy chain variable region, the T Cell Receptor (TCR) beta chain constant region extracellular section, the transmembrane region and the intracellular tail end, the linker, the antibody light chain variable region, the T Cell Receptor (TCR) alpha chain constant region extracellular section, the transmembrane region and the intracellular tail end are arranged in sequence; or the like, or, alternatively,

(3) the antibody light chain variable region, the T Cell Receptor (TCR) alpha chain constant region extracellular section, the transmembrane region and the intracellular tail end, the linker, the antibody heavy chain variable region, the T Cell Receptor (TCR) beta chain constant region extracellular section, the transmembrane region and the intracellular tail end are sequentially arranged; or the like, or, alternatively,

(4) the antibody light chain variable region, the T Cell Receptor (TCR) beta chain constant region extracellular section, the transmembrane region and the intracellular tail end, the linker, the antibody heavy chain variable region, the T Cell Receptor (TCR) alpha chain constant region extracellular section, the transmembrane region and the intracellular tail end are arranged in sequence; or the like, or, alternatively,

(5) replacing the alpha chain of the T Cell Receptor (TCR) in the steps (1) to (4) with the gamma chain of the T Cell Receptor (TCR), and replacing the beta chain of the T Cell Receptor (TCR) with the nucleic acid corresponding to the delta chain of the T Cell Receptor (TCR).

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 chain and/or β chain 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 chain and/or β chain of the TCR are subjected to cysteine point mutations. Specifically, the 48 th amino acid of the TCR alpha chain constant region is mutated into cysteine, and the 57 th amino acid of the TCR beta chain constant region is mutated into cysteine, so that the first peptide chain and the second peptide chain are connected in a manner of adding a disulfide bond.

More preferably, the nucleotide sequence of the human TCR alpha chain constant region cysteine mutant is SEQ ID NO 1, and the amino acid sequence is SEQ ID NO 11; the nucleotide sequence of the humanized TCR beta 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 first amino acid of the constant region of the natural human TCR alpha chain exists in four forms; the polypeptide with Asp as the first amino acid and Asn, His or Tyr as the first amino acid, so that the human TCR alpha chain constant region cysteine mutant may be also selected from the polypeptide expressed with the following sequence:

the amino acid sequence is SEQ ID NO. 31, and the corresponding nucleotide sequence is SEQ ID NO. 25;

the 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 TCR alpha 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 murine TCR beta constant region cysteine mutant is SEQ ID NO. 8, and the amino acid sequence is SEQ ID NO. 18.

Furthermore, the first amino acid of the constant region of the natural murine TCR alpha chain exists in four forms; the first amino acid is Asn, namely the polypeptide shown in SEQ ID NO. 17, and the polypeptide of which the first amino acid is Asp, His or Tyr, so that the murine TCR alpha chain constant region cysteine mutant can also be selected from the polypeptides represented by the following sequences:

the amino acid sequence is SEQ ID NO. 34, and the corresponding nucleotide sequence is SEQ ID NO. 28;

35 is an amino acid sequence, and 29 is a corresponding nucleotide sequence;

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) α chain can be replaced with a T Cell Receptor (TCR) γ chain; the T Cell Receptor (TCR) β chain may be replaced by a T Cell Receptor (TCR) δ chain. Preferably, the T Cell Receptor (TCR) gamma chain and the T Cell Receptor (TCR) delta chain may be derived from human TCR gamma chain or delta chain constant regions, and may also be derived from murine TCR gamma chain or delta chain constant regions.

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 alpha constant region (C alpha), and the second polypeptide is an antibody light chain variable region (V alpha)_L) And TCR beta 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 alpha constant region (C alpha)_L) And TCR beta constant region (C)_β) Fusion, or antibody light chain variable region (V)_L) Antibody heavy chain variable region (V) fused to TCR alpha constant region (C alpha)_H) And TCR beta constant region (C)_β) Fusing; and the relative furin and P2A front-to-back order of the two exchanges. The light chain and heavy chain variable regions of the antibody may be replaced with antibody variable regions of various specificities, such as anti-EGFR, CD19, CD20, and the like. A variety of variants of TCR α and β constant regions may 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.

Although the CAR-T therapy is frequent, the safety problem is a key concern in the industry and FDA, and the avoidance of self-activation to reduce side effects is an urgent problem to be solved at present. The invention introduces the antibody antigen binding fragment to be fused with the original TCR to form the chimeric T cell receptor (STAR) combined by the antibody-T cell receptor, further improves the pairing of an alpha chain and a beta 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 acid, reduces the occurrence risk of side effect and improves the safety. The result of the verification of chimeric T cell receptors of multiple targets shows that the chimeric T cell receptors 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 equivalent, more importantly, the chimeric T cell receptors have no self-activation phenomenon under the resting state without antigen stimulation, and the CAR has high self-activation phenomenon.

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:

FIG. 1 schematic structural diagrams of STAR and CAR molecules. After being expressed in cells, STAR is connected by two polypeptide chains 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 alpha constant region (Calpha), and the second polypeptide chain is formed by fusing an antibody light chain variable region (VL) and a TCR beta constant region (Cbeta); STAR functions as a complex with the CD3 subunits (epsilon, delta, lambda, zeta) endogenously expressed by T cells.

FIG. 2 shows the structural sequence of the STAR and CAR molecules. The STAR gene sequence is connected through polypeptide fragments of furin and P2A protease cutting sites, the two polypeptide chains are transcribed and translated together to be expressed into protein, and then the protein is cut into two independent proteins by furin and protease corresponding to P2A. There are various combinations of STAR: an antibody heavy chain variable region (VH) is fused with a TCR alpha constant region (ca), an antibody light chain variable region (VL) is fused with a TCR beta constant region (cp), or an antibody light chain variable region (VL) is fused with a TCR alpha constant region (ca), an antibody heavy chain variable region (VH) is fused with a TCR beta constant region (cp); and the relative furin and P2A front-to-back order of the two exchanges.

Figure 3 upper membrane situation of EGFR targeted 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 stained with anti-human TCR-. alpha./β flow antibody, followed by flow assay. It was found that STAR was stained with anti-TCR- α/β antibodies at a level comparable to native E1-TCR, compared to non-transgenic negative control cells. This result indicates that the STAR molecule can be filmed and its alpha and beta chains can be paired.

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. Jurkat Clone 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/mL IL-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-human CD69-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-human CD69-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 the α and β chains of the TCR in STAR were derived from cDNA of human peripheral blood T cells by PCR molecular cloning; on the basis of the original TCR sequence, the 48 th and 57 th amino acid sites of the constant regions of the alpha chain and the beta chain are respectively mutated into cysteine to help form an additional disulfide bond between the alpha chain and the beta chain and increase the mutual pairing efficiency, and the TCR is named 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, a first polypeptide chain fused to the TCR β chain and a second polypeptide chain fused to the α chain. The gene sequence of STAR is connected by polypeptide fragments of furin and p2A protease cutting sites, two polypeptide chains are transcribed and translated together into a fusion polypeptide, and then are cut into two independent protein subunits by furin and p2A corresponding proteases, the two subunits are covalently bonded by disulfide bonds, and form a complex with the endogenous CD3 subunit (epsilon, delta, gamma, zeta) of the T cell (as shown in fig. 1 and fig. 2).

The whole gene is inserted into a lentivirus expression vector pHAGE through restriction enzyme cutting sites NheI and NotI. The vector carries ampicillin resistance, the EF1 alpha promoter and the IRES-RFP fluorescent reporter gene.

4. Cloning and Assembly of Gene fragments

The four fragments "Cetux VL", "TCR beta-C", "Cetux-VH" and "TCR alpha-C" were obtained by cloning from pHAGE-Cetux-28zCAR vector and pHAGE-E1-TCR vector, respectively. Each pair of primers carries 25bp bases homologous to the front and back, and the four fragments are recombined and connected to a lentiviral vector in one step by a Gibson Assembly method. Thus obtaining STAR.

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

the nucleotide sequence of the E1 TCR beta 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 DH5 alpha strain and allowed to grow overnight on LB plates containing benzyl amine. Selecting the monoclonal bacterium for sequencing, wherein the sequencing primer selects the primers seq-pHAGE-F and seq-pHAGE-R on the pHAGE carrier.

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 after 3 days of infection were stained with a flow antibody against anti-human TCR α/β -BV421, followed by flow detection. It was found (fig. 3) that STAR could be stained by anti-TCR α/β antibodies compared to non-transgenic negative control cells, and at a level comparable to native E1-TCR. This result indicates that the STAR molecule can be filmed and its alpha and beta chains can 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 from human peripheral blood T cells or mouse spleen T cells is obtained by PCR molecular cloning; on the basis of the original TCR sequence, the 48 th and 57 th amino acid sites of the constant regions of the alpha chain and the beta chain are respectively mutated into cysteine to help form an additional disulfide bond between the alpha chain and the beta chain and increase the mutual pairing efficiency, and the amino acid sites are respectively named as E1-TCR (human source) or E11-TCR (mouse source).

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 fused to the TCR β chain as a first polypeptide chain and FMC63-VH fused to the α chain as a second polypeptide chain. The gene sequence of STAR is connected by polypeptide fragments of furin and p2A protease cutting sites, the two polypeptide chains are transcribed and translated together into a fusion polypeptide, and then are cut into two independent protein subunits by furin and protease corresponding to p2A, the two subunits are covalently bonded by disulfide bonds and form a complex with the endogenous CD3 subunit (epsilon, delta, gamma, zeta) of the T cell.

4. Cloning and Assembly of Gene fragments

The obtained four fragments, namely 'FMC 63-VL', 'TCR beta-C', 'FMC 63-VH' and 'TCR alpha-C', are cloned from pHAGE-FMC63-41BBzCAR vector and pHAGE-E1-TCR vector respectively. Each pair of primers carries 25bp bases homologous to the front and back, and the four fragments are recombined and connected to a lentiviral vector in one step by a Gibson Assembly method. Thus obtaining 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, OFA-VL fused to the TCR β chain as a first polypeptide segment and OFA-VH fused to the α chain as a second polypeptide segment. The gene sequence of STAR is connected by polypeptide fragments of furin and p2A protease cutting sites, the two polypeptide chains are transcribed and translated together into a fusion polypeptide, and then are cut into two independent protein subunits by furin and protease corresponding to p2A, the two subunits are covalently bonded by disulfide bonds and form a complex with the endogenous CD3 subunit (epsilon, delta, gamma, zeta) of the T cell.

3. Cloning and Assembly of Gene fragments

The obtained four fragments "OFA-VL", "TCR beta-C", "OFA-VH" and "TCR alpha-C" were cloned from pHAGE-OFA-41BBzCAR vector and pHAGE-E11-TCR vector, respectively. Each pair of primers carries 25bp bases homologous to the front and back, and the four fragments are recombined and connected to a lentiviral vector in one step by a Gibson Assembly method. Thus obtaining STAR.

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

the nucleotide sequence of the E11 TCR beta 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

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 can then be stimulated for 48-72 hours in a culture dish coated with anti-CD 3/CD28 antibodyT cells were observed to grow in size, to grow in aggregates, and to polarize in shape. 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 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:

wherein the antibody heavy chain variable region or antibody light chain variable region is from IMCC225 (Cetuximab, Cetuximab/Cetux), Ofatumumab (Ofatumumab, OFA), or CD19 monoclonal antibody FMC 63;

when the first subunit of the T cell receptor is a TCR α chain, the second subunit of the T cell receptor is a TCR β chain; or, when the first subunit of the T cell receptor is a TCR β chain, the second subunit of the T cell receptor is a TCR α chain; wherein the content of the first and second substances,

(1) the amino acid sequence of the TCR α chain constant region is SEQ ID NO:11,

the amino acid sequence of the TCR β chain constant region is SEQ ID NO: 12; or the like, or, alternatively,

(2) the amino acid sequence of the TCR α chain constant region is SEQ ID NO:17,

the amino acid sequence of the TCR β chain constant region is SEQ ID NO: 18;

and the 85 th amino acid of the constant region of the TCR alpha chain is mutated into alanine, and the 88 th amino acid of the constant region of the TCR beta chain is mutated into glycine,

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

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

3. The chimeric T-cell receptor according to claim 1,

the amino acid sequence of the Cetux VH is SEQ ID NO:13,

the amino acid sequence of the Cetux VL is SEQ ID NO: 14;

the amino acid sequence of the OFA-VH is SEQ ID NO:19,

the amino acid sequence of the OFA-VL is SEQ ID NO: 20;

the amino acid sequence of FMC63-VH is SEQ ID NO:15,

the amino acid sequence of FMC63-VL is SEQ ID NO: 16.

4. the chimeric T-cell receptor according to claim 1, wherein the target antigen-associated disease is cancer or a disease associated with a viral infection.

5. The chimeric T cell receptor according to claim 4, 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 or thyroid cancer; 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), or Hepatitis C Virus (HCV).

6. The chimeric T-cell receptor according to any one of claims 1 to 5, wherein said first and second peptide chains form a complex with the endogenous CD3 subunits (epsilon, delta, gamma, zeta) of the T-cell.

7. A complex formed by a chimeric T cell receptor that specifically binds to a target antigen, wherein the chimeric T cell receptor of any one of claims 1-6 is complexed with a CD3 subunit (epsilon, delta, gamma, zeta) endogenously expressed by T cells and which mediates a T cell-associated signal transduction pathway upon activation by the target antigen.

8. A nucleic acid encoding the chimeric T-cell receptor according to any one of claims 1 to 6 or the first and second peptide chains in the complex according to claim 7.

9. A nucleic acid encoding a sequence comprising:

(3) the antibody light chain variable region, the T Cell Receptor (TCR) alpha chain constant extracellular section, the transmembrane region and the intracellular tail end, the linker, the antibody heavy chain variable region, the T Cell Receptor (TCR) beta chain constant extracellular section, the transmembrane region and the intracellular tail end are sequentially arranged; or the like, or, alternatively,

(4) the antibody light chain variable region, the T Cell Receptor (TCR) beta chain constant region extracellular section, the transmembrane region and the intracellular tail end, the linker, the antibody heavy chain variable region, the T Cell Receptor (TCR) alpha chain constant region extracellular section, the transmembrane region and the intracellular tail end are arranged in sequence;

wherein the nucleic acid encodes a sequence in which the antibody heavy chain variable region and the T Cell Receptor (TCR) first subunit constant region comprise a first peptide chain; the variable region of the antibody light chain and the constant region of the second subunit of the T cell receptor form a second peptide chain;

when the first subunit of the T cell receptor is a TCR α chain, the second subunit of the T cell receptor is a TCR β chain; or, when the first subunit of the T cell receptor is a TCR β chain, the second subunit of the T cell receptor is a TCR α chain;

the antibody heavy chain variable region or the antibody light chain variable region is from IMCC225 (Cetuximab, Cetuximab/Cetux), Ofatumumab (Ofatumumab, OFA) or CD19 monoclonal antibody FMC 63;

the amino acid sequence of the TCR α chain constant region is SEQ ID NO:11 and the amino acid sequence of the TCR β chain constant region is SEQ ID NO: 12; alternatively, the amino acid sequence of the TCR α chain constant region is SEQ ID NO:17 and the amino acid sequence of the TCR β chain constant region is SEQ ID NO:18, and the 85 th amino acid of the constant region of the TCR α chain is mutated to alanine, and the 88 th amino acid of the constant region of the TCR β chain is mutated to glycine.

10. The nucleic acid of claim 9, wherein the first peptide chain and the second peptide chain are bound by a disulfide bond after expression in a T cell.

11. A vector comprising a nucleic acid sequence encoding the chimeric T-cell receptor according to any one of claims 1 to 6 or the first and second peptide chains of the complex according to claim 7, or comprising a nucleic acid according to any one of claims 8 to 10.

12. The vector of claim 11, 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.

13. An effector cell expressing a chimeric T-cell receptor according to any one of claims 1 to 6 or a complex according to claim 7 on the cell surface thereof.

14. The effector cell of claim 13, wherein the effector cell is a T cell.

15. A pharmaceutical composition comprising the chimeric T cell receptor of any one of claims 1-6, the complex of claim 7, the nucleic acid of any one of claims 8-10, the vector of any one of claims 11-12, or the effector cell of any one of claims 13-14, and a pharmaceutically acceptable carrier.

16. A kit comprising the chimeric T cell receptor of any one of claims 1-6, the complex of claim 7, the nucleic acid of any one of claims 8-10, the vector of any one of claims 11-12, the effector cell of any one of claims 13-14, or the pharmaceutical composition of claim 15.

17. Use of a chimeric T cell receptor according to any one of claims 1 to 6, a complex according to claim 7, a nucleic acid according to any one of claims 8 to 10, a vector according to any one of claims 11 to 12 or an effector cell according to any one of claims 13 to 14 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.

18. The use according to claim 17, wherein the target antigen-associated disease is a cancer or a disease associated with a viral infection, 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 or thyroid cancer; 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), or Hepatitis C Virus (HCV).

19. 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 13-14, wherein the chimeric T cell receptor specifically binds to the target antigen, and wherein the method is not a method of treatment of a disease.