CN114729305A

CN114729305A - Chimeric antigen receptor for immunotherapy, preparation method and application thereof

Info

Publication number: CN114729305A
Application number: CN202080062749.2A
Authority: CN
Inventors: 朱滨; 戴卫国
Original assignee: Suzhou Danluo Pharmaceutical Co ltd
Current assignee: Suzhou Danluo Pharmaceutical Co ltd
Priority date: 2019-10-17
Filing date: 2020-10-16
Publication date: 2022-07-08
Also published as: WO2021073624A1

Abstract

The invention discloses a chimeric antigen receptor for immunotherapy, a preparation method and application thereof. Encoding nucleic acids, expression vectors, host cells, and pharmaceutical compositions and methods of using the same for treating various diseases, such as cancer, are specifically disclosed.

Description

Chimeric antigen receptor for immunotherapy, preparation method and application thereof

Technical Field

The invention relates to the field of immunotherapy, and relates to a chimeric antigen receptor for immunotherapy, a preparation method and application thereof.

Background

A T cell (hereinafter referred to as "CAR-T cell") expressing a chimeric antigen receptor (hereinafter referred to as "CAR") refers to a recombinant T cell that: wherein a gene encoding a receptor recognizing a cancer cell surface antigen specifically expressed on the surface of a cancer cell is introduced into the T cell to kill the cancer cell. Phd.Zelig Fshhar et al, a chemist and immunologist of the Weizmann Institute of Science in Israel, successfully prepared T cells with chimeric antigen receptors by obtaining the theory: that is, when T cells having receptors that bind to antigens specifically expressed in cancer cells are artificially produced, an immune response only against cancer cells occurs, thereby killing cancer cells, and this fact was subsequently reported on PNAS in 1989.

However, early production of CAR-T cells, i.e. first generation CAR-T cells, using CD3 ζ alone as the signaling domain, also suffers from short duration, in addition to its insignificant therapeutic effect. Accordingly, efforts have been made to improve CAR-T cell responsiveness, and the result is the generation of second generation CAR-T cells in which the costimulatory domain (CD28 or CD137/4-1BB) and CD3 ζ produced are combined, wherein the number of CAR-T cells present in vivo is significantly increased compared to the number of first generation CAR-T cells. Meanwhile, second generation CAR-T cells use one type of co-stimulatory domain, while CAR-T cells that use two types of co-stimulatory domain are referred to as third generation CAR-T. Most recent research has focused on second and third generation CAR-T cells. Meanwhile, as for a method of treating cancer using CAR-T cells, it is reported that when cytotoxic T cells transformed to recognize CD19 were injected into 3 patients with chronic lymphocytic leukemia (CCL), leukemia of two patients was completely treated and the disease lasted for about 10 months (N.Engl J Med 2011; 365: 725-. CAR-T as used herein corresponds to the second generation, using 4-1BB as the costimulatory domain and CD3 ζ as the signaling domain. The antigen binding domain of CAR-T cells recognizes CD19 found on the surface of leukemic cancer cells as an antigen.

In addition, it has been reported that 27 out of 30 patients completely remitted, 67% of all patients completely remitted for 2 years, and 78% survived for 2 years when patients with acute leukemia were treated by administration of CTL 019. This result is very surprising given that the subject patients were relapsed or refractory patients (N Engl j Med 2014; 371: 1507-.

Currently, clinical trials for various hematologic cancers such as lymphoma (lymphoma), myeloma (myelomas), etc. have been conducted for therapeutic approaches using various CAR-T cells, and CAR-T is expected to be a commercially available drug. Since cancer treatment using CAR-T cells is an autologous method, the product cannot be produced on a large scale; however, this is a patient-specific treatment and therefore the high therapeutic effect is not comparable to that of the existing anticancer drugs.

Disclosure of Invention

In one embodiment, the invention provides a chimeric antigen receptor against a TCR comprising an antigen binding domain of interest, a transmembrane domain, a T cell activation signaling region. The antigen of interest is a TCR.

In the present specification, "Chimeric Antigen Receptor (CAR)" refers to a chimeric protein comprising an antigen binding domain of interest, a transmembrane domain, and a T cell activation signaling region. The chimeric protein refers to a protein comprising sequences derived from two or more different proteins. The CAR is not limited to containing only the above 3 regions, but may contain other regions.

The CAR of this embodiment comprises a target antigen binding domain of which the target antigen is a TCR.

By "antigen-binding domain of interest" is meant an extracellular region that binds an antigen of interest extracellularly when the T cell expresses the CAR. CAR expressed in CAR-T cells is transferred to the cell membrane, and the target antigen binding domain located outside the cell and the T cell activation signaling region located inside the cell are linked via a transmembrane domain that penetrates the cell membrane. When the CAR-T cell is contacted with a cell having the antigen of interest as a membrane antigen, the antigen-binding domain of interest binds to the antigen of interest, whereby a T cell activation signal is transmitted from the T cell activation signaling region into the T cell, and the T cell is activated.

The antigen binding domain of interest in the embodiments of the present invention is not particularly limited as long as it can specifically bind to a TCR, and preferably includes an antigen binding region of a monoclonal antibody (hereinafter, also referred to as "anti-TCR antibody") capable of specifically binding to a TCR. The "antigen binding Region" of an antibody refers to a Region involved in antigen binding in an antibody, and specifically, refers to a Region comprising Complementarity Determining Regions (CDRs). The antigen binding region of an antibody comprises at least one CDR of the antibody. In a preferred mode, the antigen binding region of an antibody comprises all 6 CDRs of the antibody. The CDR can be determined by any definition known as the definition of CDR, for example, the definition of Kabat, Chothia, AbM, and contact can be used. Preferably, the CDRs defined by Kabat are mentioned.

The anti-TCR antibody that can be used in the target antigen-binding region is not particularly limited, and may be a known antibody or a newly produced antibody. When an anti-TCR antibody is newly produced, the production of the anti-TCR antibody may be performed by a known method. For example, a method of obtaining a hybridoma by immunizing an animal with TCR, a phage display method, and the like can be used.

As examples of the anti-TCR antibody, there can be mentioned a peptide having SEQ ID NO: 7 as a heavy chain Variable (VH) region, having the amino acid sequence of SEQ ID NO: 8 as a light chain Variable (VL) region. The amino acid sequence of SEQ ID NO: the VH region having the amino acid sequence described in 7 has the Kabat-defined CDR1-3 amino acid sequences shown in SEQ ID NO: 1-3. In addition, the peptide consisting of SEQ ID NO: the amino acid sequences of the CDRs 1-3 defined by Kabat in the VL region consisting of the amino acid sequence described in SEQ ID NO: 4-6.

In a preferred manner, the antigen-binding region of interest may comprise the VH region and the VL region of an anti-TCR antibody. For example, a polypeptide comprising a single chain antibody (scFv) against the VH region and VL region of a TCR antibody is a preferred example of an antigen-binding region of interest. scFv is a polypeptide in which a VH region and a VL region of an antibody are connected by a peptide linker, and is generally used as a target antigen-binding region of CAR.

When an scFv is used, a peptide linker connecting the VH region and the VL region is not particularly limited, and a peptide generally used in scFv can be used. Examples of peptide linkers include SEQ ID NO: 9, but is not limited thereto.

The VH region and VL region used in scFv can use those of an anti-TCR antibody. Preferred examples of anti-TCR antibodies are as described above. The VH region and VL region used in the scFv may have a partially modified sequence as long as they maintain the binding ability to the TCR. For example, as the scFv, a sequence described below can be preferably used:

(1) an scFv comprising a sequence consisting of SEQ ID NO: a VH region of CDR1-3 of the VH region consisting of the amino acid sequence of SEQ ID NO. 7; and comprises a sequence defined by SEQ ID NO: the VL region of CDR1-3 of the VL region consisting of the amino acid sequence according to item 8 has a binding ability to TCR.

(2) An scFv comprising a sequence consisting of SEQ ID NO: 7, and a VH region consisting of the amino acid sequence of SEQ ID NO: 8 has a binding ability to TCR in the VL region comprising the amino acid sequence described in item 8.

(3) An scFv comprising a sequence consisting of SEQ ID NO: 7, and a VH region comprising an amino acid sequence obtained by mutation of one or more amino acids in the amino acid sequence of SEQ ID NO: 8 wherein the VL region comprising an amino acid sequence obtained by mutating one or more amino acids in the amino acid sequence described in 8 has a binding ability to TCR.

(4) An scFv comprising an amino acid sequence substantially identical to SEQ ID NO: 7, a VH region comprising an amino acid sequence having a sequence identity (identity) of 95% or more to the amino acid sequence of SEQ ID NO: the VL region comprising an amino acid sequence having 95% or more sequence identity (identity) with the amino acid sequence of SEQ ID NO. 8, and having a TCR-binding ability.

In the above (1), the sequences other than the CDRs (framework sequences) are preferably the framework sequences of known human antibodies. For example, the selection can be made from a framework Sequence of amino acid Sequences of Human antibodies registered in a publicly known Sequence database such as GenBank, or an amino acid Sequence selected from a common Sequence derived from various subgroups of Human antibodies (Human Most Horolous Consensus Sequence; Kabat, Sequences of Proteins of Immunological Interest, US Dept. health and Human Services,1991, e.g., E.A.).

In the above (3), "plural" may be, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and still more preferably 2 to 5. The "mutation" may be any of deletion, substitution, addition, and insertion, or may be a combination thereof. The position of the mutation is preferably a region other than CDR1-3 (i.e., a framework region).

In the above (4), the sequence identity is not particularly limited as long as it is 95% or more, and is preferably 96% or more, more preferably 97% or more, further preferably 98% or more, further more preferably 99% or more, and particularly preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, and 99.9% or more. The sequence identity (identity) of amino acid sequences is determined by adding gaps to the portions corresponding to insertions and deletions so that 2 amino acid sequences are aligned at the most with the corresponding amino acids, and by comparing the ratio of the amino acids that are aligned with the amino acid sequences except for the gaps in the obtained alignment. The sequence identity between amino acid sequences can be determined using various identity search software known in the art. For example, a value of sequence identity of an amino acid sequence can be calculated based on an alignment obtained by the known identity search software BLASTP.

As an example of scFv, a scFv comprising SEQ ID NO: 9; comprises the amino acid sequence of SEQ ID NO: 9 and has a binding ability to a TCR, wherein the polypeptide comprises an amino acid sequence in which one or more amino acids in the amino acid sequence set forth in fig. 9 are mutated; or comprises a sequence identical to SEQ ID NO: 9, which has a sequence identity (homology) of 95% or more, and has a binding ability to a TCR. The terms "plurality" and "variation" are the same as those described above. The "sequence identity" is also the same as described above.

The "transmembrane domain" refers to a region that exists through the cell membrane and connects an extracellular region and an intracellular region when a T cell expresses a CAR. The transmembrane domain is not particularly limited as long as it is a polypeptide having a function of penetrating a cell membrane. The transmembrane domain may be derived from a native protein or may be designed artificially. The transmembrane domain derived from a natural protein can be obtained from any membrane-bound protein or membrane-penetrating protein. In a preferred form, the transmembrane domain is capable of transmitting an activation signal to the T cell activation signaling region in response to binding of a target antigen relative to the target antigen binding domain.

Examples of the transmembrane domain include transmembrane domains such as an α chain and a β chain of a T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR. The organism from which these proteins are derived is not particularly limited, and is preferably a human. The amino acid sequences of these proteins can be obtained from a known sequence database such as GenBank.

The transmembrane domain is often linked to an extracellular hinge region, which refers to the region linking the antigen-binding region of interest outside the cell to the transmembrane domain.

In a preferred embodiment, the CAR of the present embodiment comprises an extracellular hinge region.

The extracellular hinge region is not particularly limited as long as it is a region capable of linking the target antigen-binding region to the transmembrane domain. Can be derived from natural proteins or can be designed artificially. The extracellular hinge region may be composed of, for example, about 1 to 100 amino acids, preferably about 10 to 70 amino acids. Preferably, the extracellular hinge region does not interfere with the TCR binding ability of the antigen binding region of interest and does not interfere with signaling from the T cell activation signaling region.

Examples of the extracellular hinge region include extracellular hinge regions such as CD8, CD28, and CD 4. Alternatively, the hinge region of an immunoglobulin (e.g., IgG 4) may be used. The organism from which the protein is derived is not particularly limited, and is preferably a human. The amino acid sequences of these proteins can be obtained from a known sequence database such as GenBank.

The extracellular hinge region and transmembrane domain may be variants of the extracellular hinge region and transmembrane domain derived from the above-mentioned natural protein, and examples thereof include the following.

(1) A polypeptide having a membrane-penetrating ability, which comprises an amino acid sequence having a sequence identity (identity) of 95% or more with the amino acid sequences derived from the extracellular hinge region and the transmembrane domain of a natural protein (for example, SEQ ID NO: 11).

(2) A polypeptide which is composed of an amino acid sequence in which one or more amino acids are varied in the amino acid sequences derived from the extracellular hinge region and transmembrane domain of a natural protein (for example, SEQ ID NO: 11), and which has membrane penetration ability.

In the above (1), the sequence identity is not particularly limited as long as it is 95% or more, but is preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more, and particularly preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, and 99.9% or more.

In the above (2), "plural" may be, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 4, and further preferably 2 or 3. The "mutation" may be any of deletion, substitution, addition, and insertion, or may be a combination thereof.

The T cell activation signaling region "refers to a region that is located intracellularly when the T cell expresses the CAR, and transmits a T cell activation signal into the T cell. In T cells, when the MHC-peptide complex is bound to a T cell Receptor (T cell Receptor: TCR), a T cell activation signal is transmitted to the cell through the TCR. multidot. CD3 complex, and various phosphorylation signals are caused (primary signaling). In addition, it is known that co-stimulatory molecules expressed on the cell surface of T cells transmit co-stimulatory signals into the cells by binding to ligands specific to the respective co-stimulatory molecules expressed on the cell surface of antigen presenting cells, thereby assisting in the activation of T cells (secondary signaling).

In the present specification, "T cell activation signaling" includes both the aforementioned primary signaling and secondary signaling. The "T cell activation signaling region" refers to an intracellular region of a protein involved in the primary signaling and the secondary signaling, which is involved in the signaling.

The T cell activation signaling region is not particularly limited as long as it is a T cell activation signaling region of a protein involved in T cell activation signaling. For example, an immunoreceptor tyrosine-based activation motif (ITAM) is known to be involved in primary signaling. Therefore, an example of the T cell activation signaling region includes a T cell activation signaling region of a protein having ITAM. Examples of proteins having ITAMs include: CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD66d, and the like. The T cell activation signaling region of ITAMs comprising these proteins is a preferred example of a T cell activation signaling region for CARs. More preferred examples thereof include T cell activation signaling regions such as CD3 ζ.

Furthermore, as described above, costimulatory molecules are involved in secondary signaling. Therefore, as an example of the T cell activation signaling region, a signaling region of a co-stimulatory molecule may be mentioned. Examples of co-stimulatory molecules include CD2, CD4, CD5, CD8, CD27, CD28, OXO40(CD134), 4-1BB (CD137), ICOS, CD154, HVEM, GITR, Fc Receptor-associated γ chain, and the like. The T cell activation signaling region of these proteins is also a preferred example of a T cell activation signaling region for a CAR. More preferred examples include T cell activation signaling regions such as CD28 and 4-1 BB.

The organism from which the protein is derived is not particularly limited, and is preferably a human. The amino acid sequences of these proteins can be obtained from a known sequence database such as GenBank.

The T cell activation signaling region may be a variant of the T cell activation signaling region derived from the natural protein as described above. Examples of the variants derived from the activation signaling region of the natural protein include the following variants.

(1) A polypeptide which comprises an amino acid sequence having a sequence identity (identity) of 95% or more with an amino acid sequence derived from a T cell activation signaling region of a natural protein (for example, SEQ ID NO: 12 or 13) and which has a T cell activation signaling ability.

(2) A polypeptide which is derived from a natural protein and has a T-cell activation signaling ability, and which comprises an amino acid sequence obtained by modifying one or more amino acids in the amino acid sequence of a T-cell activation signaling region (for example, SEQ ID NO: 12 or 13).

In the above (1), the sequence identity is not particularly limited as long as it is 95% or more, but is preferably 96% or more, more preferably 97% or more, further preferably 98% or more, further more preferably 99% or more, and particularly preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, and 99.9% or more.

In the above (2), "a plurality of" may be 2 to 30, preferably 2 to 20, more preferably 2 to 10, and further preferably 2 to 5, for example, in the case of using a protein involved in primary signal transduction. In addition, for example, in the case of using costimulatory molecules, the number of "a plurality" may be 2 to 15, preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 or 3. The "mutation" may be any of deletion, substitution, addition, and insertion, or may be a combination thereof.

The CAR of the present invention may include a plurality of T cell activation signaling regions, and the number of T cell activation signaling regions is not limited to 1. The multiple T cell activation signaling regions may be the same or different. In a preferred mode, the CAR comprises more than 2T cell activation signaling regions. In this case, the CAR comprises a T cell activation signaling region that is preferably a combination of a T cell activation signaling region involved in primary signaling and a T cell activation signaling region involved in secondary signaling. Specific examples thereof include a combination of the T cell activation signaling domains of CD3 ζ and CD28, a combination of the T cell activation signaling domains of CD3 ζ and 4-1BB, and a combination of CD3 ζ, CD28 and 4-1 BB.

In the case where only one T cell activation signaling region is used, it is preferable to use a T cell activation signaling region involved in one signaling, and it is more preferable to use a T cell activation signaling region of CD3 ζ.

The CAR of the present invention may further comprise a signal peptide or the like in addition to the above-described region.

A "signal peptide" is a peptide that indicates localization of a membrane protein or a secreted protein. The signal peptide is usually a peptide consisting of about 5 to 60 amino acids present at the N-terminus of a membrane protein, and is removed from a mature protein that has been localized.

The signal peptide used in the CAR of the invention is preferably a signal peptide that directs localization to the cell membrane, preferably a signal peptide of a membrane protein. Examples of the signal peptide include signal peptides such as an α chain and a β chain of a T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, an immunoglobulin heavy chain, and an immunoglobulin light chain. Specific examples of the amino acid sequence of the signal peptide include SEQ ID NO: 10 in the sequence listing.

In a specific embodiment of the invention, the signal peptide is disposed at the N-terminus of the CAR.

The aforementioned regions of the CAR of the present invention may be arranged in the order of the antigen-binding region of interest, transmembrane domain, and T-cell activation signaling region from the N-terminus. These regions may be directly connected to each other, or may be connected to each other via another region, a spacer sequence, or the like.

In a specific example of the CAR of the present invention, the CAR is a polypeptide that is configured from the N-terminus in the order of a signal peptide, an antigen-binding region of interest, an extracellular hinge region, a transmembrane domain, a T cell activation signaling region for secondary signaling, and a T cell activation signaling region for primary signaling.

The anti-TCR CAR of the invention has an amino acid sequence identical to SEQ ID NO: 14 has at least 95% or more sequence identity to the amino acid sequence shown in seq id No. 14.

The invention also provides a cell expressing the CAR described above.

The cell is preferably a mammalian cell, for example, a human cell, or a non-human mammalian cell such as a mouse, rat, cow, sheep, horse, dog, pig, monkey, and more preferably a human cell. The type of the cells is not particularly limited, and examples thereof include cells collected from blood, bone marrow fluid, spleen, thymus, lymph node, and the like; immune cells infiltrated into cancer tissues such as primary tumor, metastatic tumor and cancerous ascites. Preferred examples thereof include immune cells, and peripheral blood mononuclear cells isolated from peripheral blood can be preferably used. Among cells contained in peripheral blood mononuclear cells, effector cells are preferable, and particularly preferable cells include T cells and their precursor cells. The type of T cell is not particularly limited, and examples thereof include any T cell such as α β T cell, γ δ T cell, CD8 positive T cell, cytotoxic T cell, CD4 positive T cell, helper T cell, memory T cell, naive T cell, tumor infiltrating T cell, natural killer T cell, and the like. Among them, CD 8-positive T cells or cytotoxic T cells are more preferable.

The cell of the present invention can be obtained by introducing a polynucleotide or vector comprising a base sequence encoding the CAR of the present invention described later into the cell.

The present invention provides a polynucleotide comprising a base sequence encoding a CAR of the present invention.

The polynucleotide of the present invention is not particularly limited as long as it contains a base sequence encoding the CAR of the present invention. The polynucleotide of the invention preferably comprises a base sequence encoding the amino acid sequence of the CAR as described above.

In a specific embodiment of the present invention, as the base sequence encoding the antigen-binding region of interest, a base sequence encoding the amino acid sequence of SEQ ID NO: 1-6. May comprise a nucleotide sequence encoding SEQ ID NO: 7 and 8, or a nucleotide sequence of the amino acid sequence. May comprise a nucleotide sequence encoding SEQ ID NO: 9 in a nucleotide sequence of the amino acid sequence described in 9.

As the base sequence encoding the extracellular hinge region and transmembrane domain, a base sequence encoding the amino acid sequence of SEQ ID NO: 11 in a nucleotide sequence of the amino acid sequence described in 11.

As the base sequence encoding the T cell activation signaling region, a base sequence encoding the nucleotide sequence of SEQ ID NO: 12 or 13, or a nucleotide sequence of the amino acid sequence.

The base sequence encoding the signal peptide may include a base sequence encoding the amino acid sequence of SEQ ID NO: 10 under stringent conditions.

As the base sequence encoding the CAR, a base sequence encoding SEQ ID NO: 14 in a nucleotide sequence of the amino acid sequence described in 14.

The nucleotide sequence encoding each of the above-mentioned regions is not limited to a known one, and may be any sequence as long as it encodes each of the above-mentioned regions. Due to the gene-encoded condensation, there are a plurality of codons corresponding to 1 amino acid. Therefore, there are a plurality of nucleotide sequences encoding the same amino acid sequence. The nucleotide sequence encoding each of the above-mentioned regions may be any of a plurality of nucleotide sequences resulting from the polycondensation of gene codes, as long as it encodes these regions. The nucleotide sequences encoding the above-mentioned regions are preferably codon-optimized according to the species of the cell to be introduced, and in the case of introduction into a human cell, human codon-optimization is preferably performed. The nucleotide sequence encoding each of the above regions may be a nucleotide sequence encoding a variant of each region derived from a natural protein.

The polynucleotide of the present invention can be obtained by linking polynucleotides comprising the nucleotide sequences encoding the regions of the CAR of the present invention directly or via a spacer. Polynucleotides encoding the regions of the CAR of the present invention can also be obtained by chemical synthesis using known methods based on the base sequence of each region. Alternatively, the polynucleotide encoding each region may be obtained by amplifying polynucleotides encoding each region by a PCR method, an isothermal amplification method, or the like using, as a template, cDNA obtained by reverse transcription of DNA extracted from T cells or the like or RNA extracted from T cells or the like. The polynucleotide encoding each region obtained in this manner may be modified by substitution, deletion, addition, insertion, or the like, as long as the function of each region after translation is not lost.

The polynucleotide of the present invention may include, in addition to the base sequence encoding the CAR of the present invention, a control sequence such as a promoter, an enhancer, a poly a addition signal, a terminator, and the like, a base sequence encoding another protein, and the like.

The invention provides a vector comprising a polynucleotide encoding a CAR of the invention.

The polynucleotide may be in the form of a vector. The type of vector is not particularly limited, and a commonly used expression vector or the like can be used. The vector may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector. Examples of the vector include a viral vector, a plasmid vector, an episomal vector, and an artificial chromosome vector.

Examples of the viral vector include Sendai virus vectors, retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Hilbes virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

Examples of the plasmid vectors include plasmid vectors for animal cell expression such as pA1-11, pXT1, pRc/CMV, pRc/RSV and pcDNAI/Neo.

Episomal vectors are vectors capable of autonomous replication outside the chromosome. Examples of episomal vectors include vectors containing a sequence required for autonomous replication derived from EBV, SV40, and the like as a vector element. Specific examples of the vector elements required for autonomous replication include a gene encoding an origin of replication and a protein that binds to the origin of replication and controls replication. For example, the EBV includes the replication origin oriP and EBNA-1 gene, and the SV40 includes the replication origin ori and SV40LT gene.

Examples of the artificial chromosome vector include a YAC (Yeast specific chromosome) vector, a BAC (bacterial specific chromosome) vector, and a PAC (P1-derived specific chromosome) vector.

A preferred example of the vector of the present invention is a viral vector, and a more preferred example is a retroviral vector. Examples of the retroviral vector include a pMSGV1 vector (Tamada k et al, Clin Cancer Res 18: 6436-6445(2012)) and a pMSCV vector (Takara Bio Inc.). By using a retrovirus vector, genes in the vector are incorporated into the genome of a host cell, and can be stably expressed in the host cell for a long period of time.

The vector of the present invention may further comprise a base sequence encoding an origin of replication, a protein which binds to the origin of replication and controls replication, a base sequence encoding a marker gene such as a drug resistance gene or a reporter gene, and the like.

The invention provides pharmaceutical compositions comprising a polynucleotide, vector or CAR expressing cell as described above.

The pharmaceutical composition of the present invention may further contain other ingredients such as a pharmaceutically acceptable carrier. Examples of the other components include, in addition to a pharmaceutically acceptable carrier, a T cell activating factor such as a cytokine, an immune activator, an immune checkpoint inhibitor, other CAR-expressing cells, an anti-inflammatory agent, and the like, but are not limited thereto. Examples of the pharmaceutically acceptable carrier include a cell culture medium, physiological saline, a phosphate buffer, a citrate buffer, and the like.

The pharmaceutical composition of the present invention can be administered to a patient by a known method, preferably by injection or infusion. The route of administration is preferably intravenous administration, but is not limited thereto, and administration may be performed by injection into a tumor or the like.

The pharmaceutical compositions of the invention can comprise a therapeutically effective amount of a CAR-expressing cell. "therapeutically effective amount" refers to an amount of the agent effective for the treatment or prevention of a disease. The therapeutically effective amount may vary depending on the state of the disease, age, sex, body weight, etc., of the subject to which it is administered. In the pharmaceutical composition of the invention, the therapeutically effective amount of the CAR-expressing cells described above can be, for example, an amount in which the CAR-expressing cells are capable of inhibiting the proliferation of a tumor.

The amount and interval of administration of the pharmaceutical composition of the present invention can be appropriately selected depending on the age, sex, body weight, etc. of the subject to be administered, the kind, degree of progression, symptoms, etc. of the disease, the method of administration, etc. The dose can be administered in a therapeutically effective amount, for example, 1X10 in 1 administration, the number of cells administered⁴～1X10 ¹⁰Preferably 1X10⁵～1X10 ⁹More preferably 5X10⁶～5X10 ⁸And (4) respectively.

The administration interval of the pharmaceutical composition of the present embodiment may be, for example, 1 week, 10 to 30 days, 1 month, 3 to 6 months, 1 year, or the like. In addition, CAR-expressing cells can autonomously proliferate in vivo in a subject to be administered, and thus can be administered at once. In addition, the number of CAR-expressing cells in vivo after administration can be monitored, and the timing of administration can be determined based on the results.

In addition, the pharmaceutical composition of the present invention may be used in combination with other anticancer agents. Examples of the other anticancer agents include alkylating agents such as cyclophosphamide, metabolic antagonists such as pentostatin, molecular targeting agents such as rituximab, kinase inhibitors such as imatinib, proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporine, anticancer and antitumor substances such as idarubicin, plant alkaloids such as irinotecan, platinum agents such as cisplatin, hormone therapy agents such as tamoxifen, and immune control drugs such as epidivorin and pembrolizumab, but the present invention is not limited thereto.

The invention provides a kit for making a CAR expressing cell comprising the vector as described above. The kit is not particularly limited as long as it contains the vector described above, and may contain instructions for producing CAR-expressing cells, reagents for introducing the vector into cells, and the like.

The present invention provides a method of immunotherapy or combating transplant rejection, said method comprising administering to a patient a polynucleotide, vector, cell or pharmaceutical composition as described above.

The patients comprise patients with autoimmune diseases and cancer patients.

The cancer patients include acute myeloid leukemia patients, chronic myeloid leukemia patients, acute lymphocytic leukemia patients, hodgkin's lymphoma patients, neuroblastoma patients, ewing sarcoma patients, multiple myeloma patients, myelodysplastic syndrome patients, BPDCN patients, glioma patients, or other solid tumor patients: including patients with pancreatic cancer, lung cancer, colorectal cancer, breast cancer, and bladder cancer.

In particularly preferred embodiments, the cancer patient is a T cell lymphoma patient (such as, for example, Anaplastic Large Cell Lymphoma (ALCL), peripheral T cell lymphoma-unspecified type (PTCL-NOS), angioimmunoblastic T cell lymphoma (AITL), and other T cell lymphomas). Preferably, the cancer is characterized by expression or overexpression of a TCR.

The graft rejection reaction comprises graft-versus-host reaction and host-versus-graft reaction.

The present invention provides an anti-cancer gene therapy method comprising administering to a patient a polynucleotide, vector, or pharmaceutical composition as described above.

The cancer patients include acute myeloid leukemia patients, chronic myeloid leukemia patients, acute lymphocytic leukemia patients, hodgkin's lymphoma patients, neuroblastoma patients, ewing sarcoma patients, multiple myeloma patients, myelodysplastic syndrome patients, BPDCN patients, glioma patients, or other solid tumor patients: including patients with pancreatic cancer, lung cancer, colorectal cancer, breast cancer, and bladder cancer. Cancer is characterized by the expression or overexpression of a TCR.

In particularly preferred embodiments, the cancer is a T cell lymphoma (such as, for example, Anaplastic Large Cell Lymphoma (ALCL), peripheral T cell lymphoma-unspecific type (PTCL-NOS), angioimmunoblastic T cell lymphoma (AITL), and other T cell lymphomas). Preferably, the cancer is characterized by expression or overexpression of a TCR.

The invention provides the use of a polynucleotide, vector as hereinbefore described in the preparation of a cell or pharmaceutical composition as hereinbefore described.

The invention provides the use of a cell as hereinbefore described in the preparation of a pharmaceutical composition as hereinbefore described.

The invention provides the use of a polynucleotide, vector, cell, or pharmaceutical composition as hereinbefore described in the manufacture of a medicament for immunotherapy.

The invention provides the use of a polynucleotide, vector, cell, or pharmaceutical composition as described above in the preparation of a medicament for the treatment of cancer.

Cancer is as defined above.

The invention provides the use of a polynucleotide, vector, cell, or pharmaceutical composition as hereinbefore described in the manufacture of a medicament for the treatment of transplant rejection.

The invention provides the use of a TCR in the preparation of a chimeric antigen receptor against the TCR.

The invention provides the use of antibodies or antigen-binding regions thereof directed against a TCR in the preparation of a chimeric antigen receptor directed against a TCR.

The chimeric antigen receptor against the TCR is as described previously.

The invention also provides a method of destroying TCR positive cells, the method comprising the steps of:

1) obtaining TCR positive cells as target cells;

2) using a cell whose cell membrane surface expresses a chimeric antigen receptor against TCR as a second cell;

3) contacting the second cell obtained in step 2 with the target cell of step 1.

Further, the chimeric antigen receptor against TCR is as defined above.

The invention also provides a method of producing an engineered cell, the method comprising the steps of:

1) obtaining immune cells from a donor;

2) inhibiting endogenous TCR expression in the immune cells obtained in step 1;

3) introducing a polynucleotide encoding a chimeric antigen receptor of a recombinant anti-TCR into the cells obtained from the treatment of step 3;

preferably, the first and second electrodes are formed of a metal,

the method further comprises the steps of: 4) introducing at least one polynucleotide encoding a recombinant chimeric antigen receptor (a chimeric antigen receptor that is not anti-TCR) into the cells obtained from the treatment of step 2;

step 3 and step 4 may be performed simultaneously, or step 3 may be performed first and step 4 may be performed second, or step 4 may be performed first and step 3 may be performed second.

Further, the donor is a healthy person and not a patient.

Further, the chimeric antigen receptor against TCR is specific for a TCR epitope, specific for an epitope of a TCR-related protein, or specific for an epitope of a TCR subunit.

Further, the polynucleotide encoding the chimeric antigen receptor of the recombinant anti-TCR comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 14 in a nucleotide sequence of the amino acid sequence described in 14.

A technique for inhibiting endogenous TCR expression in immune cells obtained in step 1 involves the introduction of mRNA encoding a rare-cutting endonuclease directed against a genomic sequence.

The rare-cutting endonuclease includes TAL effector, CRISPR CAS9, ZFN.

Endogenous TCR expression in immune cells can also be inhibited using commonly used RNA interference techniques.

The method for introducing a polynucleotide encoding a recombinant chimeric antigen receptor into the cells obtained by the treatment in step 2 is not particularly limited, and a known method can be used. Examples thereof include a virus infection method, a lipofection method, a microinjection method, a calcium phosphate method, a DEAE-dextran method, an electroporation method, a method using a transposon, and a particle gun method.

The present invention may also be produced by assembling a polynucleotide comprising a base sequence encoding a CAR into the genome of a cell so that the polynucleotide can be expressed under the control of an appropriate promoter, using a known gene editing technique or the like. Examples of the gene editing technique include a technique using an endonuclease such as a zinc finger nuclease, a TALEN (transcription activator-like effector), a crispr (clustered regulated interstitial Short Palindromic repeat) -Cas system, or a ppr (pentatricopeptide repeat).

Further, the immune cells obtained from the donor include T cells. T cells include regulatory T cells, cytotoxic T cells, helper T cells, memory T cells.

Further, the T cells include CD4+ T cells, CD8+ T cells.

The chimeric antigen receptor in step 4 above is specific for a cell surface antigen.

Cell surface antigens useful in the present invention include CAIX, ROR1, CD20, CD44v7/8, CEA, EGP-2, EGP-40, erb-B2/3/4, FBP, fetal acetylcholine receptor, EGFRvIII, BCMA, CD33, GD3, CD19, CD38, HSP70, CD30, FAP, HER2, CD79a, CD79B, CD123, CD22, CLL-1, MUC-1, GD2, O acetyl GD2, CS1, KDR, LEY, MAGE-A1, mesothelin, PSCA, PSMA, TAG-72, VEGF-R2.

The chimeric antigen receptor in step 4 above is single-chain or multi-chain.

Drawings

FIG. 1 shows a schematic diagram of LV-TCRCAR plasmid constructed according to the present invention;

FIG. 2 is a graph showing the results of measuring the transduction rate of lentiviruses by flow cytometry;

FIG. 3 is a graph showing the results of TCR knockdown using flow cytometry;

FIG. 4 is a graph showing the results of flow cytometry to detect killing of Jurkat-GFP cells by CAR-T cells;

FIG. 5 shows a graph of the results of using animal models to study the effect of CAR-T cells constructed according to the present invention on tumors;

FIG. 6 shows a statistical plot of fluorescence intensity in mice;

FIG. 7 shows a statistical plot of mouse survival time;

FIG. 8 shows a statistical plot of the effect of LV-TCRCAR-T on the clearance of TCR positive cells.

Detailed Description

The following examples further illustrate the invention. The following examples are intended to illustrate the invention and should not be construed as limiting.

Example 1 CAR expression against TCR

1. Nucleic acid molecules that synthesize anti-TCR CAR

The sequences were sequentially linked in the order of the signal peptide-ScFv-hinge region and transmembrane domain-T cell activation signaling region for secondary signaling-T cell activation signaling region for primary signaling to form a nucleic acid molecule expressing an anti-TCR CAR with the sequence number TCRCAR (SEQ ID NO: 15).

3. Construction of LV-TCRCAR expression plasmid

TCRCAR is inserted into an expression vector pLVX-Puro (the linear sequence of the vector is shown as SEQ ID NO: 16) in an enzyme digestion connection mode to construct LV-TCRCAR expression plasmids, the schematic diagram of the LV-TCRCAR plasmids is shown as figure 1 (the intracellular co-stimulatory domain is 4-1BB, EGFR D III-D VI can be used as a CAR expression detection marker and a suicide gene of a CAR-T cell, and the safety of the product is improved). Enzyme cutting site: XbaI, EcoRI. Transformation, plating, sequencing by miniprep, and confirming the success of plasmid construction. The plasmid is extracted to obtain endotoxin-free expression plasmid for packaging slow virus.

4. LV-TCRCAR lentivirus package

PEI transfection method (for T75 flasks) the procedure was as follows:

(1) day1 resuscitated 293T/17 cells to 1 × T75, medium volume 15 ml;

(2) day3 passaged 293T/17 cells to 1 × T225, medium volume 45 ml;

(3) day5 passaged 293T/17 cells to 3 × T225, at a seeding density of approximately 6 × 10⁷Individual cells/T225 flask;

(4) day6 was packaged in the afternoon. The state of the cells was observed before transfection, and transfection was performed at a confluence of about 90%. The medium in the flask was discarded and replaced with 15ml of fresh DMEM medium (without antibiotics) and incubated for 30 min.

Preparing a solution A: taking 17.7 mu g of LV-TCRCAR expression plasmid, 8.8 mu g of helper plasmid pRSV-REV, 8.8 mu g of helper plasmid pMDLg/pRRE and 4.4 mu g of helper plasmid pMD2.G, the transfection ratio is 4:2:2:1, the total amount is 40 mu g, evenly mixing, diluting with serum-free DMEM to constant volume to 0.75ml, evenly mixing, and standing at room temperature for 5 min.

Preparing a solution B: 630. mu.l of DMEM was added to 120. mu.l of PEI working solution (1mg/ml, stored at 4 ℃ C.), mixed well and allowed to stand at room temperature for 5 min.

And dropwise adding the solution B into the solution A, gently mixing uniformly, and incubating at room temperature for 20 min. The mixture was added dropwise to the cells, gently mixed, and incubated in 5% carbon dioxide for 17 h.

(5) day7 is discarded in the morning, 15ml of DMEM medium without serum and antibiotics is added, the virus is harvested after 31h of culture, and then the medium is added for 24h of culture, and the virus is harvested again. Cell supernatants were harvested and centrifuged at 2000rpm for 5 min. Then transferring the supernatant into a high-speed centrifugal tube, balancing, then centrifuging at 30000g and 4 ℃ for 4h, completely sucking the supernatant, adding 500 mu l of sterile PBS buffer solution to resuspend virus particles, uniformly mixing 200 mu l/piece, subpackaging and storing in a refrigerator at-80 ℃.

5. T cell isolation

Blood samples from healthy donors are obtained from a central blood station or hospital. Patients eligible for the following tests for the disease (not limited to these tests). The method comprises the following steps: hepatitis A, hepatitis B, hepatitis C, AIDS, syphilis antibody, tuberculosis, hereditary diseases, etc. T cells were isolated according to the protocol provided using Pan T Isolation Kit human (Order No. 130-096-535) from America and whirlpool.

6. T cell activation

Preparing a complete culture medium for the T cells: OpTsizer^TMCTS ^TMT-cell Expansion SFM+5％CTS Immune cell SR+1％L-glutamine+10ng/ml IL-7/15。

The starting cell number was 3M + Human T-Activator TCR/CD28 Dynabeads 75. mu.l. The starting cell concentration was 1M/ml. Culturing in 37 deg.C incubator. Activation was carried out for 48 hours.

7. T cell gene editing

Sgrnas were designed using CRISPR/cas9 system to electrically knock out TCRs. Cas9 protein and sgrnas were purchased from ThermFisher, inc.

The electric rotating body is:

and (3) electrotransfer conditions: 1600V, 10ms, 3pulses

Wherein, the TCR sgRNA sequence is as follows:

cagggttctggatatctgt(SEQ ID NO：17)

8. LV-TCRCAR lentivirus transduction

After 12 hours of T cell gene editing, LV-TCRCAR lentivirus was transduced, and viral MOI: 3-20, polybrene 1.5. mu.l (5-10. mu.g/ml). After 6-12 hours, the lentiviral-containing medium was removed, replaced with fresh medium, and CAR-T cell expansion was performed.

9. CAR-T cell expansion

After replacing the fresh culture medium, in the presence of IL-7/15, cell passage was carried out at an initial cell density of 1M/ml, and the cell density and the survival rate were measured every 2 days, and fresh culture medium and cytokines were supplemented. The cell density was kept at 1M/ml.

10. CAR-T cell TCR knockdown efficiency assay

After 48 hours of electrotransformation, the TCR knockdown effect was detected using a flow cytometer. As shown in FIG. 2, the TCR knockdown rate reached 80-90%, a small amount of TCR/. alpha.beta.TCR/. gamma.delta.remained in LV-TCRCAR-T (T cells transfected with LV-TCRCAR knocked out, CAR-T cells) cells, TCR-positive cells were < 1%, PanT in the figure represents untreated T cells, PanT TCRKO represents TCR knocked out T cells, and LV-TCRCAR-T represents CAR-T cells.

11. LV-TCRCAR lentivirus transduction assay

After 2-7 days of lentiviral transduction, the transduction rate was measured using a flow cytometer. As a result, as shown in FIG. 3, the expression rate of CAR was not less than 50% 3 days after lentiviral transduction, in which PanT represents untreated T cells, PanT TCRKO represents TCR knock-out T cells, and LV-TCRCAR-T represents CAR-T cells.

Example 2 CAR-T cell killing Capacity assay in vitro

1. Jurkat-GFP cell line was co-cultured with the CAR-T cells prepared in example 1 at E/T (Jurkat-GFP: CAR-T) ratios of 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1, respectively.

2. The grouping is as follows:

Jurkat-GFP group: 0.5M per well, three multiple wells;

PanT TCRKO (TCR-knocked-out T cell) group: 0.5M per well, three multiple wells;

PanT TCRKO (TCR knockout T cell) + Jurkat-GFP set: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0: 1;

LV-TCRCAR (CAR-T) + Jurkat-GFP set: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0: 1;

three multiple holes are arranged in each proportion.

3. After 24 hours the flow cytometer detected the GFP fluorescence of Jurkat-GFP cells.

4. As a result, the

As shown in FIG. 4, CAR-T (LV-TCRCAR-T) cells killed 100% of TCR/TCR positive Jurkat-GFP cells 1 day at E: T2: 1.

Example 3CAR-T cell in vivo killing function assay

One, step

1. Construction of NPG mouse tumor model by Jurkat-Fluc cell line

NPG mice 5-8 weeks old, all female, injected by tail vein at 1X10⁶Jurkat-Fluc cells. And detecting the biological fluorescence after one week to confirm that the NPG mouse tumor model is successfully constructed.

2. One week later, the NPG mice were divided into a tumor model group (negative control group), a LV-CD3CAR-T group (positive control group), and a LV-TCRCAR-T group, for three groups, each of three mice.

3. CAR positive cells 1 × 10 by NPG mouse tail vein reinfusion⁷And (4) respectively. The observation period was 8 weeks.

4. Each group of NPG mice was observed weekly for bioluminescence intensity, body weight, status, and survival time.

Second, result in

In vivo efficacy results are shown in figure 5, CAR-T group significantly inhibited tumor growth, with bioluminescence intensity significantly lower than that of tumor group. Survival time was significantly prolonged in CAR-T group mice. The experimental group mice survived the observation period. The LV-TCRCAR-T group had a better tumor suppression effect than the positive control group.

In vivo efficacy results are shown in figure 6: the fluorescence intensity of the CAR-T group mice in vivo is obviously lower than that of the tumor model group, which reflects that the tumor load of the CAR-T group is obviously lower than that of the tumor model group, and the CAR-T can effectively kill tumor cells in the mice.

In vivo efficacy results are shown in figure 7: the survival time of the CAR-T group mice is obviously superior to that of the tumor model group, which can show that the CAR-T group inhibits the growth of tumors, slows down the development of diseases and can obviously prolong the survival time of the mice.

In vitro and in vivo experiments prove that the TCR-resistant CAR-T constructed by the invention can effectively kill TCR positive cells and can be used for treating T cell-derived lymphocytic leukemia and T cell-derived lymphoma.

Example 4 functional assay of CAR-T cells to inhibit transplant rejection

One, step

1. Obtaining PBMCs of allogeneic healthy donors, extracting Pan T cells (untreated T cells) and counting;

2. cell counting to determine the number of LV-TCRCAR-T;

3. determining the conductivity of LV-TCRCAR-T by flow detection;

4. the numbers of LV-TCRCAR-T and Pan T cells were adjusted to 0.5:1,1: 1 and 2: 1;

5. and (5) flow-detecting the number of TCR positive cells at 0h, 24h and 48 h.

Second, result in

The results are shown in figure 8, CAR-T cells constructed according to the invention were effective in clearing TCR positive cells in the foreign body. Therefore, the TCRCAR-T of the invention can be used for inhibiting the occurrence of transplant rejection. In fig. 8: UCAR-T stands for LV-TCRCAR-T.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

A method of destroying a TCR positive cell, the method comprising the steps of:

1) acquiring TCR positive cells as target cells;

2) using a cell whose cell membrane surface expresses a chimeric antigen receptor against TCR as a second cell;

3) contacting the second cell obtained in step 2 with the target cell of step 1.
The method of claim 1, wherein the anti-TCR chimeric antigen receptor comprises a target antigen binding domain, a transmembrane domain, a T cell activation signaling region; the antigen binding domain of interest comprises the antigen binding region of an anti-TCR antibody.
The method of claim 1, wherein the antigen binding region of the anti-TCR antibody is a polypeptide of a single chain antibody comprising a heavy chain variable region and a light chain variable region of the anti-TCR antibody.
The method of claim 3, wherein the sequence of the single chain antibody is selected from any one of:

which comprises a polypeptide consisting of SEQ ID NO: 7 in the VH region of CDR1-3 of the VH region consisting of the amino acid sequence described in SEQ ID No. 7; and comprises a sequence defined by SEQ ID NO: 8 in the VL region of CDR1-3 of the VL region comprising the amino acid sequence of SEQ ID NO. 8, which has the ability to bind to a TCR;

which comprises a sequence represented by SEQ ID NO: 7, and a VH region consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a VL domain comprising the amino acid sequence of SEQ ID NO. 8, which has a TCR-binding ability;

which comprises the amino acid sequence represented by SEQ ID NO: 7, and a VH region consisting of an amino acid sequence obtained by mutation of one or more amino acids in the amino acid sequence described in SEQ ID NO: 8 wherein the VL region comprising an amino acid sequence in which one or more amino acids in the amino acid sequence described in 8 are mutated has a binding ability to TCR;

or which comprises a sequence identical to SEQ ID NO: 7, a VH region comprising an amino acid sequence having a sequence identity of 95% or more to the amino acid sequence of SEQ ID NO: the VL region comprising an amino acid sequence having 95% or more sequence identity to the amino acid sequence of SEQ ID No. 8, having a TCR-binding ability.
The method of claim 4, wherein the single chain antibody comprises any one of:

comprises the amino acid sequence of SEQ ID NO: 9;

comprises the amino acid sequence of SEQ ID NO: 9 and has a binding ability to a TCR, wherein the polypeptide comprises an amino acid sequence in which one or more amino acids in the amino acid sequence set forth in fig. 9 are mutated;

or comprises a sequence identical to SEQ ID NO: 9 has a sequence identity of 95% or more, and has a binding ability to a TCR.
The method of claim 2, wherein the transmembrane domain comprises the transmembrane domain of any one or more of the following molecules: the α and β chains of the T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR.
The method of any one of claims 1-6, wherein the chimeric antigen receptor against a TCR further comprises an extracellular hinge region, and the transmembrane domain is linked to the extracellular hinge region.
The method of claim 7, wherein the amino acid sequences of the extracellular hinge region and transmembrane domain comprise:

and SEQ ID NO: 11, and having a membrane-penetrating ability, wherein the polypeptide comprises an amino acid sequence having a sequence identity of 95% or more to the amino acid sequence represented by 11;

or by SEQ ID NO: 11, and has membrane-penetrating ability.
The method of claim 2, wherein the T cell activation signaling region comprises a T cell activation signaling region of an ITAM-bearing protein comprising CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD66 d; and/or

T cells comprising co-stimulatory molecules including CD2, CD4, CD5, CD8, CD27, CD28, OXO40, 4-1BB, ICOS, CD154, HVEM, GITR, Fc receptor-associated gamma chain activate the signaling region.
The method of claim 9, wherein the T cell activation signaling region comprises:

(1) consisting of SEQ ID NO: 12 or 13 has a sequence identity of 95% or more, and has a T cell activation signaling ability;

(2) consisting of SEQ ID NO: 12 or 13, and having a T cell activation signaling ability.
The method of claim 2, wherein the chimeric antigen receptor against a TCR further comprises a signal peptide, and wherein the source of the signal peptide comprises the following molecules: the alpha and beta chains of the T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, immunoglobulin heavy chain, immunoglobulin light chain.
The method of claim 11, wherein the signal peptide comprises SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.
The method of any one of claims 1-12, wherein the chimeric antigen receptor against TCR has an amino acid sequence that is identical to SEQ ID NO: 14 has at least 95% or more sequence identity with the amino acid sequence shown in seq id no.
A method of producing an engineered cell, the method comprising the steps of:

1) obtaining immune cells from a donor;

2) inhibiting endogenous TCR expression in the immune cells obtained in step 1;

3) introducing a polynucleotide encoding a chimeric antigen receptor of a recombinant anti-TCR into the cells obtained from the treatment of step 3;

preferably, the first and second electrodes are formed of a metal,

the method further comprises the steps of: 4) introducing at least one polynucleotide encoding a recombinant chimeric antigen receptor into the cells obtained by the treatment of step 2;

step 3 and step 4 may be performed simultaneously, or step 3 may be performed first and step 4 may be performed second, or step 4 may be performed first and step 3 may be performed second.
The method of claim 14, wherein the donor is a healthy person and not a patient.
The method of claim 14, wherein the chimeric antigen receptor against the TCR is specific for a TCR epitope, specific for an epitope of a TCR-associated protein, or specific for an epitope of a TCR subunit.
The method of claim 14, wherein the polynucleotide encoding the chimeric antigen receptor of a recombinant anti-TCR comprises a polynucleotide encoding a polypeptide that differs from the sequence of SEQ ID NO: 14 or a nucleotide sequence of an amino acid sequence having at least 95% sequence identity to the amino acid sequence of 14.
The method of claim 14, wherein the technique of inhibiting endogenous TCR expression in the immune cells obtained in step 1 comprises introducing mRNA encoding a rare-cutting endonuclease directed against a genomic sequence, an RNA interference technique.
The method of claim 18, wherein the rare-cutting endonuclease comprises a TAL effector, CRISPR CAS9, ZFN.
The method of claim 14, wherein introducing the polynucleotide encoding the recombinant chimeric antigen receptor into the cells obtained by the step 2 comprises using a viral vector.
The method of claim 14, wherein the immune cells obtained from the donor comprise T cells.
The method of claim 21, wherein the T cell comprises a regulatory T cell, a cytotoxic T cell, a helper T cell, a memory T cell.
The method of claim 14, wherein the chimeric antigen receptor in step 4 is specific for a cell surface antigen.
The method of claim 23, wherein said cell surface antigen comprises CAIX, ROR1, CD20, CD44v7/8, CEA, EGP-2, EGP-40, erb-B2/3/4, FBP, fetal acetylcholine receptor, EGFRvIII, BCMA, CD33, GD3, CD19, CD38, HSP70, CD30, FAP, HER2, CD79a, CD79B, CD123, CD22, CLL-1, MUC-1, GD2, O acetyl GD2, CS1, KDR, LEY, MAGE-a1, mesothelin, PSCA, PSMA, TAG-72, VEGF-R2.
The method of claim 14, wherein the chimeric antigen receptor in step 4 is single-chain or multi-chain.
An anti-TCR chimeric antigen receptor comprising an antigen-binding domain of interest, a transmembrane domain, a T cell activation signaling region; the antigen of interest is a TCR.
The chimeric antigen receptor against a TCR according to claim 26 wherein the antigen binding domain of interest comprises the antigen binding region of an anti-TCR antibody.
The anti-TCR chimeric antigen receptor of claim 26, wherein the antigen-binding region of the anti-TCR antibody is a polypeptide comprising a single chain antibody of the heavy chain variable region and the light chain variable region of the anti-TCR antibody.
A chimeric antigen receptor against a TCR as claimed in claim 28 wherein the sequence of the single chain antibody is selected from any one of:

which comprises a polypeptide consisting of SEQ ID NO: a VH region of CDR1-3 of the VH region consisting of the amino acid sequence of SEQ ID NO. 7; and comprises a sequence defined by SEQ ID NO: a VL region of CDR1-3 of the VL region consisting of the amino acid sequence according to item 8, which has TCR-binding ability;

which comprises a sequence defined by SEQ ID NO: 7, and a VH region consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a VL domain comprising the amino acid sequence of SEQ ID NO. 8, which has a TCR-binding ability;

which comprises the amino acid sequence represented by SEQ ID NO: 7, and a VH region comprising an amino acid sequence in which one or more amino acids in the amino acid sequence described in SEQ ID NO: 8 wherein the VL region comprising an amino acid sequence in which one or more amino acids in the amino acid sequence described in 8 are mutated has a binding ability to TCR;

or which comprises a sequence identical to SEQ ID NO: 7, a VH region comprising an amino acid sequence having a sequence identity of 95% or more to the amino acid sequence of SEQ ID NO: the VL region comprising an amino acid sequence having 95% or more sequence identity to the amino acid sequence of SEQ ID No. 8, having a TCR-binding ability.
The chimeric antigen receptor against a TCR according to claim 29 wherein the single chain antibody comprises any one of:

comprises the amino acid sequence of SEQ ID NO: 9;

comprises the amino acid sequence of SEQ ID NO: 9 and has a binding ability to a TCR, wherein the polypeptide comprises an amino acid sequence in which one or more amino acids in the amino acid sequence set forth in fig. 9 are mutated;

or comprises a sequence identical to SEQ ID NO: 9 has a sequence identity of 95% or more to the amino acid sequence shown in seq id No. 9, and has a binding ability to TCR.
The chimeric antigen receptor against a TCR according to claim 26 wherein the transmembrane domain comprises the transmembrane domain of any one or more of the following molecules: the α and β chains of the T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR.
A chimeric antigen receptor against a TCR according to any one of claims 26 to 31 which further comprises an extracellular hinge region to which the transmembrane domain is linked.
The chimeric antigen receptor against a TCR according to claim 32 wherein the amino acid sequences of the extracellular hinge and transmembrane domains comprise:

and SEQ ID NO: 11, and having a membrane-penetrating ability, wherein the polypeptide comprises an amino acid sequence having a sequence identity of 95% or more to the amino acid sequence represented by 11;

or by SEQ ID NO: 11, and has membrane-penetrating ability.
The chimeric antigen receptor against a TCR according to claim 26 wherein the T cell activation signaling region comprises the T cell activation signaling region of an ITAM-bearing protein including CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 e, CD5, CD22, CD79a, CD79b, CD66 d; and/or

T cells that comprise a co-stimulatory molecule including CD2, CD4, CD5, CD8, CD27, CD28, OXO40(CD134), 4-1BB (CD137), ICOS, CD154, HVEM, GITR, Fc receptor-associated gamma chain activate signaling region.
The chimeric antigen receptor against a TCR according to claim 34 wherein the T cell activation signaling region comprises:

(1) consisting of SEQ ID NO: 12 or 13, which has a sequence identity of 95% or more, and has a T cell activation signaling ability;

(2) consisting of SEQ ID NO: 12 or 13, and having a T cell activation signaling ability.
The chimeric antigen receptor against a TCR according to claim 26, further comprising a signal peptide derived from a molecule selected from the group consisting of: the alpha and beta chains of the T cell receptor, CD3 ζ, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, immunoglobulin heavy chain, immunoglobulin light chain.
The chimeric antigen receptor against a TCR according to claim 36 wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.
A chimeric antigen receptor against a TCR according to any one of claims 26 to 37 which has an amino acid sequence substantially identical to that of SEQ ID NO: 14 has at least 95% or more sequence identity to the amino acid sequence shown in seq id No. 14.
A polynucleotide comprising a base sequence encoding the chimeric antigen receptor against TCR of any one of claims 14-26.
The polynucleotide according to claim 39, wherein the base sequence encoding the antigen-binding region of interest comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1-6, or a nucleotide sequence of the amino acid sequence; or comprises a nucleotide sequence encoding SEQ ID NO: 7 or 8; or comprises a nucleotide sequence encoding SEQ ID NO: 10 in a nucleotide sequence of the amino acid sequence described in [ 10 ].
The polynucleotide according to claim 39, comprising as a base sequence encoding an extracellular hinge region and transmembrane domain a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 in a nucleotide sequence of the amino acid sequence described in 11.
The polynucleotide according to claim 39, which comprises a nucleotide sequence encoding a T cell activation signaling region represented by SEQ ID NO: 12 or 13, or a nucleotide sequence of the amino acid sequence.
The polynucleotide according to claim 39, which comprises a nucleotide sequence encoding the signal peptide represented by SEQ ID NO: 10 in a nucleotide sequence of the amino acid sequence described in [ 10 ].
The polynucleotide according to claim 39, comprising as the base sequence encoding the CAR a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 in a nucleotide sequence of the amino acid sequence described in 14.
A vector comprising the polynucleotide of any one of claims 39-44.
An engineered cell comprising the chimeric antigen receptor against a TCR according to any one of claims 26 to 38, the polynucleotide according to any one of claims 39 to 44, or the vector according to claim 45.
The cell of claim 46, wherein the source of the cell comprises a T cell.
A pharmaceutical composition comprising the polynucleotide of any one of claims 39-44, the vector of claim 45, the cell of claim 46 or 47.
The pharmaceutical composition of claim 48, further comprising a pharmaceutically acceptable carrier, T-cell activating factor, immune activator, immune checkpoint inhibitor, other CAR-expressing cells, or an anti-inflammatory agent.
A kit for making cells expressing a chimeric antigen receptor against a TCR, the kit comprising the vector of claim 45.
A method of immunotherapy comprising administering to a patient the cell of claim 46 or 47, or the pharmaceutical composition of claim 48 or 49.
The method of claim 51, wherein the patient comprises an autoimmune disease patient, a cancer patient.
The method of claim 52, wherein the cancer patient comprises an acute myeloid leukemia patient, a chronic myeloid leukemia patient, an acute lymphocytic leukemia patient, a Hodgkin's lymphoma patient, a neuroblastoma patient, an Ewing's sarcoma patient, a multiple myeloma patient, a myelodysplastic syndrome patient, a BPDCN patient, a glioma patient, or another solid tumor patient: including patients with pancreatic cancer, lung cancer, colorectal cancer, breast cancer, and bladder cancer.
A method of combating transplant rejection comprising administering to a patient the cell of claim 46 or 47, or the pharmaceutical composition of claim 48 or 49.
The method of claim 54, wherein said transplant rejection response comprises a graft versus host response, a host versus graft response.
An anti-cancer gene therapy comprising administering to a patient the polynucleotide of any one of claims 39-44, the vector of claim 45, or the pharmaceutical composition of claim 48 or 49.
The method of claim 56, wherein the patient comprises an acute myeloid leukemia patient, a chronic myeloid leukemia patient, an acute lymphocytic leukemia patient, a Hodgkin's lymphoma patient, a neuroblastoma patient, an Ewing's sarcoma patient, a multiple myeloma patient, a myelodysplastic syndrome patient, a BPDCN patient, a glioma patient, or another solid tumor patient: including patients with pancreatic cancer, lung cancer, colorectal cancer, breast cancer, and bladder cancer.
Use of the polynucleotide of any one of claims 39-44, or the vector of claim 45, in the preparation of the cell of claim 46 or 47.
Use of the polynucleotide of any one of claims 39-44, or the vector of claim 45, or the cell of claim 46 or 47, for the preparation of a pharmaceutical composition of claim 48 or 49.
Use of the polynucleotide of any one of claims 39-44, or the vector of claim 45, in the preparation of a kit of claim 50.
Use of the polynucleotide of any one of claims 39-44, or the vector of claim 45, the cell of claim 46 or 47, or the pharmaceutical composition of claim 48 or 49, for the manufacture of a medicament for the treatment of a disease, wherein the disease comprises an autoimmune disease, cancer.
The use of claim 61, wherein the cancer comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, Hodgkin's lymphoma, neuroblastoma, Ewing's sarcoma, multiple myeloma, myelodysplastic syndrome, BPDCN, glioma, or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.
Use of the polynucleotide of any one of claims 39-44, or the vector of claim 45, the cell of claim 46 or 47, or the pharmaceutical composition of claim 48 or 49, for the preparation of a medicament for the treatment of transplant rejection.
Use of the polynucleotide of any one of claims 39-44, or the vector of claim 45, the cell of claim 46 or 47, or the pharmaceutical composition of claim 48 or 49 in the manufacture of a medicament for immunotherapy.
Use of a TCR for the preparation of a chimeric antigen receptor against the TCR.
Use of an antibody or antigen-binding region thereof directed against a TCR in the preparation of a chimeric antigen receptor against the TCR.
The use of claim 65 or 66, wherein the chimeric antigen receptor against TCR comprises the chimeric antigen receptor against TCR of any of claims 26-38.