CN117567595B

CN117567595B - MAGE-A4 specific T cell receptor and uses thereof

Info

Publication number: CN117567595B
Application number: CN202410066664.1A
Authority: CN
Inventors: 韩研妍; 储君君; 欧淑丹; 马民骏; 吴世凡
Original assignee: Henry Is Source Of Biological Science And Technology Co ltd Shanghai; Heng Ruiyuan Zheng Guangzhou Biotechnology Co ltd
Current assignee: Henry Is Source Of Biological Science And Technology Co ltd Shanghai; Heng Ruiyuan Zheng Guangzhou Biotechnology Co ltd
Priority date: 2024-01-17
Filing date: 2024-01-17
Publication date: 2024-05-14
Anticipated expiration: 2044-01-17
Also published as: CN117567595A

Abstract

The present invention provides a tumor-specific T cell receptor that specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex, engineered T cells comprising the receptor, and methods of using the same for treating diseases including cancer.

Description

MAGE-A4 specific T cell receptor and uses thereof

Technical Field

The present application relates to the field of cancer immunotherapy. In particular, the application relates in some aspects to binding molecules, such as complexes capable of specifically binding Major Histocompatibility Complex (MHC) molecules and MAGE-A4 peptides. In particular, the application relates to T Cell Receptors (TCRs), including antigen binding domains thereof, that bind to or recognize such peptide epitopes. The application further relates to engineered cells comprising such binding molecules, e.g., TCRs, and their use in adoptive cell therapies.

Background

The human body possesses a complex immune system that protects itself from diseases, including in vivo malignancies. The innate immune response to tumors is usually elicited by tumor antigens, including muteins that are expressed exclusively in cancer cells, as well as tumor-associated antigens (TAAs) that are overexpressed in tissues of cancer origin but are not recognized as "self" at all. Antigen Presenting Cells (APCs), particularly Dendritic Cells (DCs), that encounter tumor antigens can process and present tumor antigens to their cell surfaces. After maturation, DCs loaded with tumor antigens can elicit T cell responses to cancer cells hosting tumor antigens, involving cytotoxic T cells, helper T cells, and functionally different effector T cells and memory T cells. Cytotoxic T cells can specifically bind to and recognize cancer cells expressing tumor antigens through their surface T Cell Receptors (TCRs) and kill cancer cells by releasing cytokines, enzymes and cytotoxins or by triggering pro-apoptotic signaling cascades via cell-cell interactions.

Adoptive lymphocyte therapy (ACT) provides a promising concept for cancer treatment by administering ex vivo expanded lymphocytes to a subject. For example, tumor-infiltrating lymphocytes (TILs) may be isolated from a patient's tumor, the TILs expanded ex vivo, and the TILs infused back into the patient after removal of the patient's natural non-myeloid lymphocytes. However, variability in TIL affinity isolated from each patient or donor limits the anti-tumor efficacy in clinical trials. Furthermore, most antigen-specific mutations that lead to an immune response are found only in the cancer of an individual, not in multiple patients.

An engineered T cell containing a tumor antigen specific T Cell Receptor (TCR) can overcome some of the challenges faced by current methods of adoptive lymphocyte therapy, as it can rapidly produce tumor-reactive T lymphocytes with defined antigen specificity. There is a need in the art to obtain TCRs that target different tumor antigens and that provide high anti-tumor efficacy and low off-target effects in vivo. Problems with common TCR-T cell therapies include serious adverse events in clinical trials (such as central nervous system toxicity), which may be associated with improper selection of targets (so-called on-target off tumor effect) and biased proliferation of T cell populations (biological expansion).

Melanoma-associated antigen A4 (melanoma-associated antigen A4, MAGE-A4) is one of the members of the MAGE protein family of tumor antigens, which in healthy tissues are expressed only in immune-free sites. MAGE-4 is generally expressed in solid tumors, including synovial sarcoma, mucoid liposarcoma, non-small cell lung cancer, head and neck squamous carcinoma, ovarian cancer, urothelial cell carcinoma, melanoma, and gastroesophageal carcinoma. MAGE-A4 specific TCR and TCR-T are therefore promising cancer therapies with high anti-tumor efficacy and low off-target effects.

Disclosure of Invention

According to a first aspect of the present invention there is provided an isolated tumour specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex, the tumour specific TCR comprising: a TCR a chain comprising CDR1 having the amino acid sequence of SEQ ID No.1, CDR2 having the amino acid sequence of SEQ ID No. 2, and CDR3 having the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 having the amino acid sequence of SEQ ID NO. 4, CDR2 having the amino acid sequence of SEQ ID NO. 5 and CDR3 having the amino acid sequence of SEQ ID NO. 6.

In some embodiments, an isolated tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a major tissue compatibility complex (MHC)/MAGE-A4 epitope complex is provided, wherein the MAGE-A4 epitope comprises the amino acid sequence shown in SEQ ID No. 93.

In some embodiments according to any of the above, the MHC is HLA-A x 11:01.

In some embodiments according to any one of the above, the isolated tumor-specific TCR, or antigen-binding domain thereof, comprises: a TCR alpha chain comprising a variable region having the amino acid sequence of SEQ ID NO. 7 and a TCR beta chain comprising a variable region having the amino acid sequence of SEQ ID NO. 10.

In some embodiments according to any of the above, the isolated tumor-specific TCR, or antigen-binding domain thereof, comprises:

A TCR alpha chain comprising an amino acid sequence having SEQ ID NOs 7 and 8 or an amino acid sequence having at least 90% sequence identity thereto, and a TCR beta chain comprising an amino acid sequence having SEQ ID NOs 10 and 11 or an amino acid sequence having at least 90% sequence identity thereto.

An isolated TCR, or antigen-binding domain thereof, as described in some embodiments of any of the above, which is isolated or purified or recombinant.

An isolated TCR, or antigen-binding domain thereof, according to any of the embodiments described in some of the above, which is chimeric, humanized or human.

An isolated TCR or antigen-binding domain thereof according to any of the embodiments described above, wherein the TCR or antigen-binding domain thereof is single chain.

An isolated TCR, or antigen-binding domain thereof, according to any of the embodiments above, wherein the TCR a chain further comprises an a constant (ca) region and/or the TCR β chain further comprises a β constant (cβ) region.

An isolated TCR, or antigen-binding domain thereof, according to any of the embodiments above, wherein the cα region and the cβ region are mouse constant regions.

An isolated TCR, or antigen-binding domain thereof, according to any of the embodiments above, wherein the cα region comprises the amino acid sequence of SEQ ID nos. 22 or an amino acid sequence having at least 90% sequence identity thereto, and/or the cβ region comprises the amino acid sequence of SEQ ID nos. 25 or an amino acid sequence having at least 90% sequence identity thereto.

An isolated TCR, or antigen-binding domain thereof, as described in some embodiments of any of the above, comprising: a TCR alpha chain comprising the amino acid sequences having SEQ ID NOs 21 and 22 or an amino acid sequence having at least 90% sequence identity thereto, and a TCR beta chain comprising the amino acid sequences having SEQ ID NOs 24 and 25 or an amino acid sequence having at least 90% sequence identity thereto.

An isolated TCR, or an antigen-binding domain thereof, according to any of the embodiments above, wherein the TCR a chain and/or the TCR β chain further comprises a signal peptide.

In some embodiments, an isolated nucleic acid is provided that encodes an isolated TCR or antigen-binding domain thereof, or a TCR alpha chain or a TCR beta chain thereof, according to the embodiments of any of the above.

The isolated nucleic acid molecule of any of the embodiments above, wherein the nucleic acid sequence is codon optimized.

The isolated nucleic acid molecule of any one of the embodiments above, wherein the nucleotide sequence encoding the TCR a chain and the nucleotide sequence encoding the TCR β chain are separated by a self-cleaving peptide sequence.

The isolated nucleic acid molecule according to any of the embodiments above, wherein the self-cleaving peptide is P2A comprising the amino acid sequence shown in SEQ ID NO. 13.

In some embodiments, an isolated nucleic acid is provided that encodes the TCR a chain and the TCR β chain of the tumor-specific TCR of any one of claims 1-13.

In some embodiments, an engineered immune cell is provided comprising a tumor-specific TCR or nucleic acid encoding the same, as described in any of the embodiments above, the engineered cell being a cell line or a primary or heterologous cell obtained from a subject, preferably the cell is a human cell, including but not limited to a T cell, NK cell, macrophage, or the like.

An engineered immune cell according to any one of the embodiments above, wherein the immune cell is a T cell.

An engineered cell according to any one of the embodiments above, wherein said engineered cell comprises a cd8+ T cell.

An engineered cell according to any one of the embodiments above, wherein said engineered cell comprises a cd4+ T cell.

In some embodiments, a pharmaceutical composition is provided comprising an engineered immune cell described in any of the embodiments above, and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a method of treating a MAGE-A4 expressing disease or disorder comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any one of the embodiments described above.

The method according to any one of the embodiments above, wherein the disease or disorder is cancer.

The method according to any one of the embodiments above, wherein the cancer is selected from lung cancer, liver cancer, skin cancer, breast cancer and head and neck tumor.

The method according to any one of the embodiments above, wherein the engineered cell is an autologous cell of the subject.

The method of any one of the embodiments above, further comprising administering to the subject one or more of: immunosuppressants, therapeutic antibodies, chemotherapy, radiation therapy, surgery, or any combination thereof.

In some embodiments, the isolated tumor-specific TCR, or antigen-binding domain thereof, as described in the first aspect, comprises:

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 79.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 80.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 81.

a TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 82.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 83.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 84.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 85.

a TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No.1, CDR2 of the amino acid sequence of SEQ ID No.2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 86.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No.1, CDR2 of the amino acid sequence of SEQ ID No.2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 87.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No.1, CDR2 of the amino acid sequence of SEQ ID No.2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 88.

A TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No.1, CDR2 of the amino acid sequence of SEQ ID No.2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 89.

Drawings

The following examples are intended to be purely exemplary of the present patent application and therefore should not be considered as limiting the invention in any way. The following examples and detailed description are provided by way of illustration and not by way of limitation.

FIG. 1A shows three exemplary MAGE-A4 specific TCRs which recognize antigen and present MHC of antigen; FIG. 1B shows that the invention shows MAGE-A4 specific TCR constructs (wild type, murine, and murine codon optimized constructs).

FIGS. 2A-2C show the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G 02.

FIG. 3 shows HLA restriction assays of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 in different tumor cell lines.

FIG. 4 shows the characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G 02.

FIG. 5 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive NCI-H520 cells in vitro.

FIG. 6 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 12.

FIG. 7 shows the characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 12.

FIG. 8 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM12 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive NCI-H520 cells in vitro.

FIG. 9 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 17.

FIG. 10 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 17.

FIG. 11 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM17 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive NCI-H520 cells in vitro.

FIG. 12 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 26.

FIG. 13 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 26.

FIG. 14 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM26 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive NCI-H520 cells in vitro.

FIG. 15 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 31.

FIG. 16 shows the characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 31.

FIG. 17 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM31 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive NCI-H520 cells in vitro.

FIG. 18 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 40.

FIG. 19 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 40.

FIG. 20 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM40 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive NCI-H520 cells in vitro.

FIG. 21 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 41.

FIG. 22 shows the characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 41.

FIG. 23 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM41 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive A375 cells in vitro.

FIG. 24 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 43.

FIG. 25 shows the characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 43.

FIG. 26 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM43 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive A375 cells in vitro.

FIG. 27 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 54.

FIG. 28 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 54.

FIG. 29 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM54 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive A375 cells in vitro.

FIG. 30 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 57.

FIG. 31 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 57.

FIG. 32 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM57 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive A375 cells in vitro.

FIG. 33 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 60.

FIG. 34 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 60.

FIG. 35 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM60 were cytotoxic to HLA-A 11:01 and MAGE-A4 protein double positive A375 cells in vitro.

FIG. 36 shows the selection and characterization of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM 81.

FIG. 37 shows the characterization of T cells expressing MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM 81.

FIG. 38 shows that T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02-AM81 were cytotoxic to H1299 cells which were double positive for HLA-A 11:01 and MAGE-A4 proteins in vitro.

I. definition of the definition

Before describing the present invention in detail with respect to some preferred embodiments thereof, the following general definitions are provided. Unless otherwise defined as follows, the terms are generally used herein as in the art.

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims, which do not exclude other elements when the term "comprising" is used in the description and in the claims. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter to include at least a certain number of embodiments, it should also be understood that a group consisting of preferably only these embodiments is disclosed.

For the purposes of the present invention, the term "obtained" is considered to be the preferred embodiment of the term "available". If, in the following, for example, an antibody is defined as being obtainable from a particular source, this is also to be understood as disclosing antibodies obtained from that source.

As used herein, "T cell receptor" or "TCR" refers to an endogenous or engineered T cell receptor that includes an extracellular antigen-binding domain that binds to a specific epitope in an MHC molecule. TCRs may include TCR alpha polypeptide chains and TCR beta polypeptide chains. "tumor-specific TCR" refers to a TCR that specifically recognizes a tumor antigen expressed by a tumor cell. "TCR-T" refers to T cells that express a recombinant TCR. An "antigen binding domain" of a TCR refers to a polypeptide that binds to an antigen with a higher degree of specificity due to its primary, secondary or tertiary sequence and/or post-translational modification and/or charge. The antigen binding domain may be derived from any portion or fragment of a TCR that retains the antigen binding activity of the TCR as part of the TCR. Antigen binding portions encompass, for example, portions of a TCR that retain the ability to specifically bind to the amino acid sequence of MAGE-A4 or a fragment thereof, or to detect, treat, or prevent cancer to a similar extent, to the same extent, or to a greater extent than a wild-type TCR. The TCR antigen-binding domain can comprise, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of a full-length TCR. The antigen binding domain may comprise an antigen recognition portion of either or both the alpha and beta chains of the TCR, such as a portion comprising one or more of the complementarity determining regions CDR1, CDR2 and CDR3 of the alpha and/or beta chain variable region of the TCR. The antigen binding domain may comprise the following amino acid sequence: CDR1 of the α chain, CDR2 of the α chain, CDR3 of the α chain, CDR1 of the β chain, CDR2 of the β chain, CDR3 of the β chain, or any combination thereof. Preferably, the antigen binding domain comprises the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain or the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain of the TCR; or the amino acid sequences of CDR1, CDR2, CDR3 of all alpha and beta chains. In one embodiment of the invention, the antigen binding portion may comprise, for example, the variable region of a TCR, which comprises a combination of the CDR regions described above. In this regard, the antigen binding portion may comprise the amino acid sequence of the variable region of the α chain of the TCR (vα), the amino acid sequence of the variable region of the β chain (vβ), or both vα and vβ. In one embodiment of the invention, the antigen binding portion may comprise a combination of a variable region and a constant region. In this regard, the antigen binding portion may comprise the full length alpha or beta chain of the TCR, or both the alpha and beta chains.

As used herein, the terms "specifically bind to," "recognize," "specifically recognize," "target," or "specific for … …" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, or between a receptor and a ligand, or between a receptor and an epitope/MHC complex, that determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a TCR that binds or specifically binds to an epitope of interest is a TCR that binds to an epitope/MHC complex of interest with greater affinity, binding, more readily, and/or for a longer duration than it binds to other epitope/MHC complexes. In one embodiment, the extent of binding of the TCR to the unrelated epitope/MHC complex is less than about 10% of the binding of the TCR to the epitope/MHC complex of interest, as measured (e.g., by Radioimmunoassay (RIA)). In certain embodiments, a TCR that specifically binds to an epitope of interest (i.e., an epitope of interest/MHC complex) has a dissociation constant (Kd) of 1 [ mu ] M, 100nM, 10 nM, 1 nM, or 0.1 nM. In certain embodiments, the TCR specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but need not be, exclusive binding.

II MAGE-A4 tumor specific TCR

The present application provides TCRs that specifically recognize tumor MAGE-A4 and/or specifically recognize MHC/MAGE-A4 epitope complexes. In some embodiments, the tumor-specific TCR specifically recognizes MAGE-A4. In some embodiments, the tumor-specific TCR specifically recognizes the MHC/MAGE-A4 epitope complex. Nucleic acids and vectors encoding tumor-specific TCRs, engineered immune cells expressing tumor-specific TCRs are also within the scope of the present application.

Exemplary TCRs identified using the methods described herein are shown in table 1 below. The V, J, C section is named according to IMGT database. Other nomenclature and segmentation algorithms known in the art may be used. See, e.g., lefranc, m. -p., the Immunologist, 7, 132-136 (1999), world wide web. IMGT. Org/IMGTSCIENTIFICCHART/document/IMGT-frcddefined. Html. CDR1, CDR2, and CDR3 of an exemplary TCR are shown in the "sequence listing" section.

Table 1: exemplary tumor-specific TCRs

In some embodiments, a MAGE-A4 tumor specific TCR or antigen binding domain thereof is provided that specifically binds to an epitope of a MAGE-A4 peptide, such as a MAGE-A4 epitope comprising the amino acid sequence of SEQ ID NO. 101. In some embodiments, the MAGE-A4 tumor specific TCR or antigen binding domain thereof specifically binds to a MAGE-A4 epitope comprising the amino acid sequence of SEQ ID NO. 101. In some embodiments, a MAGE-A4 tumor specific TCR or antigen binding domain thereof is provided that specifically binds to a MAGE-A4 peptide/MHC complex wherein MAGE-A4 comprises the MAGE-A4 epitope of the amino acid sequence of SEQ ID NO: 101. In some embodiments, the MHC protein in the MAGE-A4 peptide/MHC complex is an MHC-class I protein. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, HLA-A is HLA-A x 11:01. In some embodiments, the target antigen of an effector cell (e.g., T cell) that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on the surface is a MAGE-A4 _146-154 peptide/mhc i a 11:01 complex.

In some embodiments, there is provided a MAGE-A4 tumor specific TCR or antigen binding domain thereof comprising the amino acid sequence of any one of SEQ ID NOs 3, 6, 17, 20, 31, 34, 79-85. In some embodiments, there is provided a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, comprising: (a) A TCR a CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID No. 3; and a TCR beta CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID No. 6; (b) A TCR a CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID No. 17; and a TCR beta CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID No. 20; (c) A TCR a CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID No. 31; and a TCR.beta.CDR 3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 34; (d) A TCR beta CDR3 having at least about 90% sequence identity (e.g., 100% identity) to any one of SEQ ID NOs 79-89. Wherein the MAGE-A4 tumor specific TCR comprising the amino acid sequence set forth in SEQ ID NO. 79-89 or antigen binding domain thereof has a higher affinity.

In some embodiments, there is provided a MAGE-A4 tumor specific TCR or antigen binding domain thereof comprising a TCR alpha chain comprising CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO 9, 23, or 37.

In some embodiments, there is provided a MAGE-A4 tumor specific TCR or antigen binding domain thereof comprising a TCR β chain comprising CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO 12, 26, or 40.

In some embodiments, there is provided a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, comprising: a TCR alpha chain comprising CDRs of any one of the amino acid sequences of SEQ ID NOs 9, 23, or 37; and a TCR beta chain comprising the CDRs of any one of the amino acid sequences of SEQ ID NO 12, 26, or 40.

The tumor-specific TCRs described herein, or antigen binding domains thereof, also include TCR constant domains. In some embodiments, the tumor-specific TCR, or antigen-binding domain thereof, comprises: a TCR alpha chain comprising a TCR alpha constant domain (TRAC) of any of SEQ ID NOs 9, 23, 37, or a variant thereof; and a TCR β chain comprising any of the amino acid sequences of SEQ ID NOs 12, 26, 40, a TCR β constant domain (TRBC), or a variant thereof. In some embodiments, the tumor-specific TCR, or antigen-binding domain thereof, comprises: human TRAC and human TRBC, such as human C.alpha.and human C.beta.1, or human C.alpha.and human C.beta.2. In some embodiments, the tumor-specific TCR, or antigen-binding domain thereof, comprises murine TRAC and murine TRBC, such as murine cα and murine cβ1, or murine cα and murine cβ2.

In some embodiments, the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, is a human TCR. In some embodiments, the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, is a chimeric TCR, such as a murine TCR, e.g., a TCR comprising a murine constant region of the TCR a chain and the β chain. In some embodiments, the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, comprises a human TCR variable region, as well as a TCR constant region from a non-human species (such as a mouse).

In some embodiments, there is provided a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, comprising: a TCR a chain comprising an amino acid sequence that is at least about 80% identical (e.g., at least about any of 85%, 90%, 95%, 98%, or more identical, or 100% identical) to any of the amino acid sequences of SEQ ID NOs 9, 23, and 37; and a TCR β chain comprising an amino acid sequence having at least about 80% identity (e.g., any of at least about 85%, 90%, 95%, 98%, or more identity, or having 100% identity) to any of the amino acid sequences of SEQ ID NOs 12, 26, and 40.

Also provided is an isolated nucleic acid encoding the TCR alpha chain and/or the TCR beta chain of a tumor-specific TCR or an antigen-binding domain thereof according to any of the tumor-specific TCRs or antigen-binding domains thereof described above; a vector comprising the isolated nucleic acid(s). In some embodiments, the isolated nucleic acid is a murine nucleic acid sequence. In some embodiments, the isolated nucleic acid is a sequence-optimized murine nucleic acid sequence.

Immune cells expressing MAGE-A4 tumor specific TCR or antigen binding domain thereof

One aspect of the present application provides an engineered immune cell comprising a tumor-specific TCR or antigen-binding domain thereof, an isolated nucleic acid, or a vector according to any of the above.

The present application provides engineered immune cells expressing MAGE-A4 tumor specific TCRs or antigen binding domains thereof. In some embodiments, an effector cell (e.g., T cell) is provided that presents any of the MAGE-A4 tumor specific TCRs or antigen binding domains thereof according to the disclosure on its surface. In some embodiments, the effector cell comprises a nucleic acid encoding a MAGE-A4 tumor-specific TCR, or an antigen binding domain thereof, wherein the MAGE-A4 tumor-specific TCR, or antigen binding domain thereof, is expressed from the nucleic acid and localized to the surface of the effector cell. In some embodiments, the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, is exogenously expressed and combined with effector cells. In some embodiments, the effector cell is a T cell. In some embodiments, the effector cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

Cell modifications for disrupting gene expression include any such techniques known in the art including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR or TALEN based gene knockout), and the like. For example, in some embodiments, an effector cell (e.g., T cell) is provided comprising a nucleic acid encoding any of the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof described herein, wherein the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof are expressed from the nucleic acid and localized to the surface of the effector cell. In some embodiments, an effector cell (e.g., T cell) is provided that comprises a first nucleic acid sequence encoding a first polypeptide chain of a MAGE-A4 tumor-specific TCR or antigen-binding domain thereof and a second nucleic acid sequence encoding a second polypeptide chain of a MAGE-A4 tumor-specific TCR or antigen-binding domain thereof. In some embodiments, the nucleic acid encoding a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, is on a vector. In some embodiments, the first nucleic acid sequence is located on a first vector and the second nucleic acid sequence is located on a second vector. In some embodiments, the first and second nucleic acid sequences are located on the same vector. The vector may be selected from, for example, mammalian expression vectors and viral vectors (e.g., vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses). In some embodiments, the vector is integrated into the host genome of the effector cell. In some embodiments, the nucleic acid sequence encoding a MAGE-A4 tumor specific TCR or antigen binding domain thereof is under the control of a promoter. In some embodiments, the first nucleic acid sequence is under the control of a first promoter and the second nucleic acid sequence is under the control of a second promoter. In some embodiments, the first and second promoters have the same sequence. In some embodiments, the first and second promoters have different sequences. In some embodiments, the first and second nucleic acids are under the control of a single promoter. In some embodiments, the first, second, and/or single promoter is an inducible promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the expression of the first polypeptide chain is substantially the same as the expression of the second polypeptide chain. In some embodiments, the expression of the first polypeptide chain is at least about two (e.g., at least about any of 2, 3,4, 5, or more) times greater than the expression of the second polypeptide chain. In some embodiments, the expression of the first polypeptide chain is no more than about 1/2 (e.g., no more than about any of 1/2, 1/3, 1/4, 1/5, or less) of the expression of the second polypeptide chain.

Expression of a MAGE-A4 tumor specific TCR or antigen binding domain thereof can be determined from mRNA or protein levels. The expression level of mRNA can be determined by measuring the amount of mRNA transcribed from the nucleic acid using various well known methods including Northern immunoblotting, quantitative RT-PCR, microarray analysis, and the like. Protein expression levels can be measured by known methods, including immunocytochemistry staining, enzyme-linked immunosorbent assay (ELISA), western blot analysis, luminescence analysis, mass spectrometry, high performance liquid chromatography, high pressure liquid chromatography-tandem mass spectrometry, and the like.

In some embodiments, a MAGE-A4 tumor-specific TCR or antigen binding domain thereof effector cell (e.g., a T cell) is provided that expresses any of the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof described herein on its surface, wherein the MAGE-A4 tumor-specific TCR or antigen binding domain thereof effector cell comprises a host genome-integrated mammalian expression vector and viral vector (e.g., vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses) comprising a first nucleic acid sequence encoding a first polypeptide chain of the MAGE-A4 tumor-specific TCR or antigen binding domain thereof and a second nucleic acid sequence encoding a second polypeptide chain of the MAGE-A4 tumor-specific TCR or antigen binding domain thereof. In some embodiments, there is a promoter operably linked to the 5' end of the first nucleic acid sequence, and there is a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A, T2A, E a or F2A) to link the 3' end of the first nucleic acid sequence to the 5' end of the second nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are transcribed into a single RNA under the control of the promoter. In some embodiments, there is a promoter operably linked to the 5' end of the second nucleic acid sequence, and there is a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (e.g., P2A, T2A, E a or F2A) to link the 3' end of the second nucleic acid sequence to the 5' end of the first nucleic acid sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence are transcribed into a single RNA under the control of the promoter. In some embodiments, the 2A peptide is P2A. In some embodiments, the promoter is an inducible promoter. In some embodiments, the effector 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, an effector cell (e.g., a T cell) is provided that expresses a MAGE-A4 tumor specific TCR on a surface, comprising a) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID No. 9 and b) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID No. 12; and/or c) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO. 23 and d) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO. 26; and/or e) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO. 37 and f) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO. 40. In some embodiments, effector cells expressing a MAGE-A4 tumor specific TCR or antigen binding domain thereof further comprise a P2A nucleic acid sequence encoding SEQ ID NOs: 13, 27, 41. In some embodiments, an effector cell (e.g., T cell) is provided that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on a surface comprising a nucleic acid sequence encoding the amino acid sequence of one or more of SEQ ID NOs 14, 28, 42.

In some embodiments, the target antigen of an effector cell (e.g., T cell) that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on the surface is a MAGE-A4 peptide/MHC complex. In some embodiments, the MHC protein in the MAGE-A4 peptide/MHC complex is an MHC-class I protein. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, HLA-A is a 11:01. In some embodiments, the effector cell is a γδ T cell. In some embodiments, the effector cell is an αβ T cell. In some embodiments, the effector 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, the target antigen of an effector cell (e.g., T cell) that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on the surface is a MAGE-A4 _146-154 peptide/mhc i a 11:01 complex. In some embodiments, the immune cell is a T cell. In some embodiments, a pharmaceutical composition is provided that includes an engineered immune cell according to any of the engineered immune cells described above, and a pharmaceutically acceptable carrier.

Preparation of effector cells expressing MAGE-A4 TCR or antigen binding Domain thereof

In some embodiments, in one aspect, the invention provides effector cells (e.g., lymphocytes, e.g., T cells) that express a MAGE-A4 tumor-specific TCR, or an antigen binding domain thereof. Provided herein are exemplary methods of making effector cells (e.g., MAGE-A4 tumor-specific TCR effector cells, e.g., MAGE-A4 tumor-specific TCRT cells) that express a MAGE-A4 tumor-specific TCR or antigen binding domain thereof. In some embodiments, a MAGE-A4 tumor-specific TCR effector cell (e.g., a MAGE-A4 tumor-specific TCRT cell) can be produced by introducing into the effector cell one or more nucleic acids (including, for example, lentiviral vectors) encoding a MAGE-A4 tumor-specific TCR or antigen binding domain thereof (e.g., any of the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof described herein) that specifically bind to a target antigen (e.g., a disease-associated antigen). The introduction of one or more nucleic acids into an effector cell may be accomplished using techniques known in the art, such as those described herein for nucleic acids. In some embodiments, the MAGE-A4 tumor-specific TCR effector cells of the invention (e.g., MAGE-A4 tumor-specific TCRT cells) are capable of replication in vivo, resulting in long-term persistence that can facilitate sustained control of a disease (e.g., cancer) associated with expression of a target antigen.

In some embodiments, the invention relates to the use of lymphocyte infusion to administer genetically modified T cells that express a MAGE-A4 tumor specific TCR, or any of its antigen binding domains, according to the disclosure, to treat a patient suffering from or at risk of suffering from a disease and/or disorder associated with expression of a target antigen (also referred to herein as a "target antigen positive" or "TA positive" disease or disorder), including, for example, cancer. In some embodiments, autologous lymphocyte infusion is used in the treatment. Autologous PBMCs are collected from the patient in need of treatment and T cells are activated and expanded using methods described herein and known in the art and then infused back into the patient. In some embodiments, a T cell (also referred to herein as a "MAGE-A4 tumor-specific TCRT cell") is provided that expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen according to any of the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof described herein. The MAGE-A4 tumor-specific TCRT cells of the invention can undergo robust in vivo T cell expansion and can establish target antigen-specific memory cells that persist in blood and bone marrow for long periods of time at high levels. In some embodiments, MAGE-A4 tumor specific TCRT cells of the invention infused into a patient can deplete cells presenting a target antigen in vivo in a patient with a target antigen-associated disease, e.g., cancer cells presenting the target antigen. In some embodiments, the MAGE-A4 tumor-specific TCRT cells of the invention infused into a patient can eliminate in vivo cells presenting a target antigen, such as cancer cells presenting a target antigen, in a patient having a target antigen-related disorder that is difficult to treat with at least one known therapy.

Prior to T cell expansion and genetic modification, a T cell source is obtained from an individual. T cells can be obtained from a variety of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T cell lines available in the art may be used. In some embodiments of the invention, T cells may be obtained from blood units collected from an individual using any number of techniques known to those skilled in the art (e.g., ficoll ™ isolation). In some embodiments, cells from the circulating blood of the individual are obtained by blood cell separation (apheresis). The blood cell separation product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by blood cell separation may be washed to remove plasma fractions and placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. As will be readily appreciated by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, for example, by using semi-automated "flow-through" centrifugation (e.g., cobe 2991 cell processor Baxter CytoMate or Haemonetics cell holder 5) according to manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca ²⁺ -free, mg ²⁺ -free PBS, plasmaLyteA or other physiological saline solution with or without buffers. Alternatively, the undesired components of the blood cell separation sample may be removed and the cells resuspended directly in culture medium.

In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the erythrocytes, e.g., by gradient centrifugation through PERCOLL ™ or by elutriation of depleted monocytes by countercurrent centrifugation. Specific subsets of T cells (e.g., cd3+, cd28+, cd4+, cd8+, cd45ra+ and cd45ro+ T5 cells) can be further isolated by positive selection or negative selection techniques. For example, in some embodiments, T cells are isolated by incubating with anti-CD 3/anti-CD 28 (i.e., 3X 28) bound beads (e.g., DYNABEADS cube M-450CD3/CD 28T) for a period of time sufficient to positively select for the desired T cells. In some embodiments, the period of time is about 30 minutes. In some embodiments, the period of time is in the range of 30 minutes to 36 hours or longer (including all ranges between these values). In some embodiments, the period of time is at least 1, 2, 3,4, 5, or 6 hours. In some embodiments, the period of time is from 10 to 24 hours. In some embodiments, the incubation period is 24 hours. Regarding the isolation of T cells from patients with leukemia, the use of longer incubation times (e.g., 24 hours) can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where fewer T cells are present than other cell types, such as isolating Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or immunocompromised individuals. In addition, the use of longer incubation times can increase the efficiency of cd8+ T cell capture. Thus, by only shortening or extending the time allowed for T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the bead to T cell ratio, T cell subpopulations can be preferentially selected for or against other time points at the beginning of the culture or during the process. In addition, by increasing or decreasing the ratio of anti-CD 3 and/or anti-CD 28 antibodies on the beads or other surfaces, T cell subsets can be preferentially selected for or against at the beginning of culture or at other desired time points. Those skilled in the art will recognize that multiple selection wheels may also be used in the context of the present invention. In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. The "unselected" cells may also undergo other selection rounds.

Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers specific for the negative selection cells. One approach is to sort and/or select cells via negative magnetic immunoadhesion or flow cytometry using a monoclonal antibody mixture directed against cell surface markers present on negatively selected cells. For example, to enrich for cd4+ cells by negative selection, monoclonal antibody mixtures typically include antibodies directed against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In some embodiments, it may be desirable to enrich for or forward select regulatory T cells that normally express cd4+, cd25+, cd62lhi, gitr+ and foxp3+. Or in some embodiments, T regulatory cells are depleted by anti-CD 25 binding beads or other similar selection methods.

To isolate a desired cell population by positive or negative selection, the cell concentration and surface (e.g., particles, such as beads) may be varied. In some embodiments, it may be desirable to significantly reduce the volume of the beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, in some embodiments, a concentration of about 20 hundred million cells/ml is used. In some embodiments, a concentration of about 10 hundred million cells/ml is used. In some embodiments, greater than about 100,000,000 cells/ml are used. In some embodiments, a cell concentration of any of about 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, or 50,000,000 cells/milliliter is used. In some embodiments, a cell concentration of any of about 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, or 100,000,000 cells/ml is used. In some embodiments, a concentration of about 125,000,000 or about 150,000,000 cells/ml is used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest (e.g., CD28 negative T cells) or from samples where many tumor cells are present (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may be of therapeutic value and are desirably available. For example, the use of high cell concentrations allows for more efficient selection of cd8+ T cells that typically have weaker CD28 expression.

In some embodiments of the invention, the T cells are obtained directly from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, particularly after treatment with drugs that disrupt the immune system, the quality of the T cells obtained may be optimal or its ability to expand ex vivo is improved shortly after treatment during the period in which the patient will normally resume the therapy. Also, enhanced transplantation and in vivo expansion of these cells may be in a preferred state after ex vivo manipulation using the methods described herein. Thus, the context of the present invention encompasses the collection of blood cells (including T cells), dendritic cells, or other cells of the hematopoietic lineage during this recovery phase. Furthermore, in some embodiments, movement (e.g., movement with GM-CSF) and modulation schemes may be used to establish conditions in an individual, wherein it is desirable to re-proliferate, re-circulate, regenerate and/or expand specific cell types, particularly during a defined time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system. Whether before or after genetically modifying T cells to express a desired MAGE-A4 tumor specific TCR or antigen binding domain thereof, T cells can generally be activated and expanded using methods such as those described, for example, in: U.S. patent No. 6,352,694;6,534,055;6,905,680;6,692,964;5,858,358;6,887,466;6,905,681;7,144,575;7,067,318;7,172,869;7,232,566;7,175,843;5,883,223;6,905,874;6,797,514;6,867,041; U.S. patent application publication number 20060121005.

In general, T cells of the invention are expanded by contact with a surface to which are attached agents that stimulate a CD3/TCR complex-associated signal and ligands that stimulate costimulatory molecules on the surface of the T cell. In particular, the T cell population may be stimulated, for example, by contact with an anti-CD 3 antibody or antigen binding domain thereof, or an anti-CD 2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) that is bound to a calcium ionophore. To co-stimulate the accessory molecules on the surface of the T cells, ligands that bind the accessory molecules are used. For example, a population of T cells may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. anti-CD 3 antibodies and anti-CD 28 antibodies in order to stimulate proliferation of cd4+ T cells or cd8+ T cells. Examples of anti-CD 28 antibodies include 9.3, B-T3, XR-CD28 (DiaClone, besan ç on, france) and may be used in general by other methods as are generally known in the art (Berg et al, transplantProc.30 (8): 3975-3977,1998; haanen et al, J.exp. Med.190 (9): 13191328,1999; garland et al, J.Immunol meth.227 (1-2): 53-63,1999).

Genetic modification

In some embodiments, a MAGE-A4 tumor-specific TCR effector cell of the invention (e.g., a MAGE-A4 tumor-specific TCRT cell) is produced by transducing the effector cell (e.g., a T cell prepared by a method described herein) with a viral vector encoding a MAGE-A4 tumor-specific TCR as described herein. Viral vector delivery systems include DNA and RNA viruses that have episomal or integrated genomes after delivery to effector cells. For a review of gene therapy procedures, see Anderson, science256:808-813 (1992); nabel and Feigner, TIBTECH11:211-217 (1993); mitani and Caskey,TIBTECH11:162-166(1993);Dillon, TIBTECH11:167-175(1993);Miller, 5Nature357:455-460(1992);VanBrunt, Biotechnology6(10):1149-1154(1988);Vigne,RestorativeNeurology andNeuroscience 8:35-36(1995);Kremer and Perricaudet, british Medical Bulletin (l): 31-44 (1995); and Yu et al GENE THERAPY1:13-26 (1994). In some embodiments, the viral vector is a lentiviral vector, and the MAGE-A4 tumor specific TCR effector cell comprises a lentiviral vector integrated into the genome of the MAGE-A4 tumor specific TCR effector cell. In some embodiments, the MAGE-A4 tumor-specific TCR effector cell is a MAGE-A4 tumor-specific TCRT cell comprising a lentiviral vector integrated into its genome. In some embodiments, the MAGE-A4 tumor specific TCR effector cells are T cells modified to block or reduce expression of one or both of the endogenous TCR chains. For example, in some embodiments, the MAGE-A4 tumor-specific TCR effector cells are αβt cells modified to block or reduce expression of TCR α and/or β chains, or the MAGE-A4 tumor-specific TCR effector cells are γδ T cells modified to block or reduce expression of TCR γ and/or δ chains. Cell modifications for disrupting gene expression include any such techniques known in the art including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR or TALEN based gene knockout), and the like.

In some embodiments, the CRISPR/Cas system is used to generate MAGE-A4 tumor specific TCRT cells with reduced expression of one or both of the T cell's endogenous TCR chains. For a review of gene-edited CRISPR/Cas systems, see, e.g., jianW and MARRAFFINI LA, annu. Rev. Microbiol.69,2015; hsuPD et al, cell, 157 (6): 1262-1278,2014; and O' ConnellMR et al, nature516:263-266,2014. In some embodiments, MAGE-A4 tumor specific TCRT cells that produce reduced expression of one or both of the T cell's endogenous TCR chains are edited using a TALEN-based genome.

Enrichment

In some embodiments, a method of enriching a heterogeneous population of cells for MAGE-A4 tumor-specific TCR effector cells according to any of the MAGE-A4 tumor-specific TCR effector cells described herein is provided. Specific subsets of MAGE-A4 tumor-specific TCR effector cells (e.g., MAGE-A4 tumor-specific TCRT cells) that specifically bind to a target antigen can be enriched by forward selection techniques. For example, in some embodiments, MAGE-A4 tumor-specific TCR effector cells (e.g., T cells expressing a MAGE-A4 tumor-specific TCR) are enriched by incubating with beads that bind to a target antigen for a period of time sufficient to positively select for the desired MAGE-A4 tumor-specific TCR effector cells. In some embodiments, the period of time is about 30 minutes. In some embodiments, the period of time is in the range of 30 minutes to 36 hours or longer (including all ranges between these values). In some embodiments, the period of time is at least 1,2, 3,4, 5, or 6 hours. In some embodiments, the period of time is from 10 to 24 hours. In some embodiments, the incubation period is 24 hours. With respect to isolation of MAGE-A4 tumor specific TCR effector cells present at low levels in heterogeneous cell populations, the use of longer incubation times (e.g., 24 hours) can increase cell yield. Longer incubation times may be used to isolate MAGE-A4 tumor-specific TCR effector cells in any situation where there are fewer MAGE-A4 tumor-specific TCR effector cells than other cell types.

Those skilled in the art will recognize that multiple selection wheels may also be used in the context of the present invention. To isolate a desired MAGE-A4 tumor-specific TCR effector cell population by positive or negative selection, the cell concentration and surface (e.g., particles, such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of the beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, in some embodiments, a concentration of about 20 hundred million cells/ml is used. In some embodiments, a concentration of about 10 hundred million cells/ml is used. In some embodiments, greater than about 1 hundred million cells/ml are used. In some embodiments, a cell concentration of any of about 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, or 50,000,000 cells/milliliter is used. In some embodiments, a cell concentration of any of about 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, or 100,000,000 cells/ml is used. In some embodiments, a concentration of about 125,000,000 or about 150,000,000 cells/ml is used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of MAGE-A4 tumor-specific TCR effector cells that may weakly express the MAGE-A4 tumor-specific TCR or antigen binding domain thereof.

In some of any of such embodiments described herein, the enrichment results in minimal or substantially no depletion of MAGE-A4 tumor specific TCR effector cells. For example, in some embodiments, the enrichment results in depletion of less than about 50% (e.g., less than about any of 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the MAGE-A4 tumor-specific TCR effector cells. Effector cell depletion can be determined by any means known in the art, including any means described herein. In some of any of such embodiments described herein, the enrichment results in minimal or substantially no final differentiation of MAGE-A4 tumor-specific TCR effector cells. For example, in some embodiments, the enrichment results in less than about 50% (e.g., less than about any of 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the MAGE-A4 tumor-specific TCR effector cells undergoing final differentiation. Effector cell differentiation may be determined by any means known in the art, including any means described herein.

In some of any of such embodiments described herein, the enrichment results in minimal or substantially no internalization of the MAGE-A4 tumor specific TCR or antigen binding domain thereof on the MAGE-A4 tumor specific TCR effector cells. For example, in some embodiments, the enrichment results in internalization of less than about 50% (e.g., less than about any of 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the MAGE-A4 tumor-specific TCR, or antigen binding domain thereof, on the MAGE-A4 tumor-specific TCR effector cell. Internalization of the MAGE-A4 tumor specific TCR or antigen binding domain thereof on a MAGE-A4 tumor specific TCR effector cell can be determined by any means known in the art, including any means described herein.

In some of any of the embodiments described herein, the enrichment results in increased proliferation of MAGE-A4 tumor specific TCR effector cells. For example, in some embodiments, the enrichment results in an increase in the number of MAGE-A4 tumor specific TCR effector cells by at least about 10% (e.g., any of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000% or more) after the enrichment. Thus, in some embodiments, there is provided a method of enriching a heterogeneous population of cells for MAGE-A4 tumor-specific TCR effector cells expressing a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen, comprising: a) Contacting a heterogeneous population of cells with a ligand comprising a target antigen or one or more epitopes contained therein to form a complex comprising MAGE-A4 tumor specific TCR effector cells bound to the ligand; and b) isolating the complex from the heterogeneous cell population, thereby producing a MAGE-A4 tumor specific TCR effector cell enriched cell population. In some embodiments, the ligand is immobilized to a solid support. In some embodiments, the solid support is a microparticle (e.g., a bead). In some embodiments, the solid support is a surface (e.g., the bottom of a well). In some embodiments, the ligand is labeled with a tag. In some embodiments, the tag is a fluorescent molecule, an affinity tag, or a magnetic tag. In some embodiments, the method further comprises eluting MAGE-A4 tumor specific TCR effector cells from the ligand and recovering the eluate.

In some embodiments, T cells can be enriched by FACS methods by screening for CD3 and IFN-gamma. In some embodiments, T cells can be enriched by ELISA methods by screening for IFN-gamma.

Library screening

To isolate a candidate MAGE-A4 tumor-specific TCR, or antigen-binding domain construct thereof, that is specific for a target antigen, a library of MAGE-A4 tumor-specific TCRs, or antigen-binding domains thereof (e.g., cells expressing a library of nucleic acids encoding a plurality of MAGE-A4 tumor-specific TCRs, or antigen-binding domains thereof) can be exposed to a ligand comprising the target antigen, or one or more epitopes contained therein, followed by isolation of affinity members of the library that specifically bind the ligand. In some embodiments, the ligand is immobilized on a solid support. In some embodiments, the carrier may be a bead surface, a microtiter plate, an immune tube, or any material known in the art to be suitable for such purposes.

In some embodiments, interactions occur on a labeled ligand target (e.g., biotin-labeled ligand) in solution. In some embodiments, the procedure involves one or more wash steps (panning) for removing non-specific and non-responsive library members. In some embodiments, to purify the complexes in solution, the complexes are captured by immobilization or by centrifugation. In some embodiments, the affinity members are captured on a soluble biotin-labeled ligand, followed by immobilization of the affinity complex (affinity members and ligand) on streptavidin beads. In some embodiments, the solid support is a bead. In some embodiments, the beads include, for example, magnetic beads (e.g., from Bangslaboratories, polysciences, dynal Biotech, miltenyi Biotech, or Quantum Magnetic), non-Magnetic beads (e.g., pierce and Upstatetechnology), monodisperse beads (e.g., dynal Biotech and MicroparticleGmbh), and polydisperse beads (e.g., chemagen). The use of magnetic beads is described in detail in the literature (Uhlen, M et al (1994) ADVANCES IN Biomagnetic Separation, bio Techniques press, 5 Westborough, MA). In some embodiments, the affinity member is purified by forward selection. In some embodiments, the affinity members are purified by negative selection to remove undesired library members. In some embodiments, the affinity members are purified by positive and negative selection steps.

In general, the techniques used to prepare the library constructs will be based on known genetic engineering techniques. In this regard, the nucleic acid sequence encoding the MAGE-A4 tumor specific TCR or antigen binding domain thereof in the library to be expressed is incorporated into an expression vector suitable for the type of expression system to be used. Suitable expression vectors for presentation in cells such as cd3+ cells are well known and described in the art. For example, in some embodiments, the expression vector is a viral vector, such as a lentiviral vector.

In some embodiments, a nucleic acid library is provided comprising sequences encoding a plurality of MAGE-A4 tumor specific TCRs or antigen binding domains thereof according to any one of the embodiments described herein. In some embodiments, the nucleic acid library comprises viral vectors encoding a plurality of MAGE-A4 tumor specific TCRs or antigen binding domains thereof. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, there is provided a method of screening a nucleic acid library according to any of the embodiments described herein for a sequence encoding a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, specific for a target antigen, comprising: a) Introducing the nucleic acid library into a plurality of cells such that the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, 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 the sequence encoding the MAGE-A4 tumor specific TCR or antigen binding domain thereof from the cells collected in step c) thereby identifying a MAGE-A4 tumor specific TCR or antigen binding domain thereof that is specific for the target antigen. In some embodiments, the method further comprises one or more washing steps. In some embodiments, one or more washing steps are performed between steps b) and c). In some embodiments, the plurality of cells is a plurality of cd3+ cells. In some embodiments, the ligand is immobilized on a solid support. In some embodiments, the solid support is a bead. In some embodiments, collecting the cells bound to the ligand comprises eluting the cells from the ligand bound to the solid support and collecting the eluate. In some embodiments, the ligand is labeled with a tag. In some embodiments, the tag is a fluorescent molecule, an affinity tag, or a magnetic tag. In some embodiments, collecting cells that bind to a ligand comprises isolating a complex comprising the cells and a labeled ligand. In some embodiments, the cell is dissociated from the complex.

MHC proteins

The Human Leukocyte Antigen (HLA) gene is a human version of the MHC gene. Three major MHC class I proteins involved in antigen presentation in humans are HLA-A, HLDA-B and HLA-C. In some embodiments, the antigen of interest (e.g., tumor-associated or virus-encoded antigen) and mhc class i protein. In some embodiments, the MHC class I protein is HLA-A in some embodiments. Peptides suitable for use in generating antigen binding moieties may be determined, for example, based on the presence of proteasome and immunoproteasome HLA (e.g., HLA-DRA 01: 01) binding motifs and cleavage sites, using computer predictive models known to those skilled in the art. With respect to predicting MHC binding sites, such models include, but are not limited to, proPred1 (described in more detail in Singh and Raghava, proPred: prediction20 of HLA-DR binding sites, BIOINFORMATICS (12): 1236-1237, 2001) and SYFPEITHI (see Schulter et al SYFPEITHI, Database for Searching and T-Cell EpitopePrediction. Immunoinformatics Methods in Molecular Biology, , volume 409 (1): 75-93,2007).

Once the appropriate peptide is identified, peptide synthesis can be accomplished according to protocols generally known to those skilled in the art. The peptides of the invention, due to their relatively small size, can be synthesized directly in solution or on a solid support according to known peptide synthesis techniques. Various automated synthesizers are commercially available and can be used according to known schemes. The synthesis of peptides in the solution phase has become a well-established procedure for large-scale manufacture of synthetic peptides and is thus a suitable alternative method for the preparation of the peptides of the invention (see, e.g., solid PHASE PEPTIDE SYNTHESIS, JOHNMORROW Stewart and Martin et al, Application of Almez-mediated Amidation Reactionsto Solution Phase Peptide Synthesis,Tetrahedron Letters , volume 39, pages 1517-1520, 1998).

In some embodiments, the MHC protein in the MAGE-A4 peptide/MHC complex is an MHC-class I protein. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, the target antigen of an effector cell (e.g., T cell) that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on the surface is a MAGE-A4 _146-154 peptide/mhc i a 11:01 complex.

Variants

In some embodiments, amino acid sequence variants of the MAGE-A4 TCRs provided herein or antigen binding domains thereof are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of a MAGE-A4TCR or antigen binding domain thereof. Amino acid sequence variants of the MAGE-A4TCR or antigen binding domain thereof can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the MAGE-A4TCR or antigen binding domain thereof, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding. In some embodiments, MAGE-A4TCR or antigen binding domain variants thereof having one or more amino acid substitutions are provided. Amino acid substitutions may be introduced into the relevant MAGE-A4TCR or antigen binding domain thereof and the product is screened for a desired activity, such as retention/increased antigen binding or reduced immunogenicity.

Conservative substitutions are shown in table 2 below.

Amino acids can be grouped into different classes according to common side chain characteristics:

hydrophobicity: norleucine Met, ala, val, leu, ile;

neutral hydrophilicity: cys, ser, thr, asn, gln;

Acid: asp, glu;

alkaline: his, lys, arg;

residues that affect chain orientation: gly, pro;

Aromatic: trp, tyr, phe.

Non-conservative substitutions will entail changing a member of one of these classes to another class. Exemplary substitution variants are affinity matured antibody portions, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and variant antibody portions are presented on phage and screened for a particular biological activity (e.g., binding affinity).

Table 2: conservative substitutions

Briefly, one or more CDR residues are mutated and the variant is presented on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in the MAGE-A4 TCR or antigen binding domain thereof, for example, to increase affinity. Affinity maturation achieved by construction of and reselection from secondary libraries has been described, for example, in Hoogenboom et al Methods in Molecular Biology178:1-37 (O' Brien et al, human Press, totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any TCR or antigen binding domain portion variants thereof having the desired affinity. CDR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling. Specifically, CDR3 is typically targeted.

In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody moiety to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the TCR or antigen-binding domain thereof that do not substantially reduce binding affinity. In certain embodiments of the variant TCRs or antigen binding domains thereof provided above, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

A suitable method for identifying residues or regions of an antibody moiety that can be targeted for mutation-induction is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or set of target residues (e.g., charged residues, such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the TCR or antigen-binding domain thereof with the MHC antigen complex is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-MHC-TCR portion complex can be determined to identify the point of contact between the antibody portion and the antigen. Such contact residues and neighboring residues may be targeted or excluded from the substitution candidates. Variants may be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include TCR portions with N-terminal methionyl residues. Other insertional variants of the TCR portion include fusions of the N-or C-terminus of the TCR portion with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody portion.

Derivatives and their use as inhibitors of viral infection

In some embodiments, a MAGE-A4 tumor-specific TCR, or an antigen binding domain thereof, according to any of the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof described herein, can be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for MAGE-A4 tumor specific TCRs or antigen binding domain derivatives thereof include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, poly-5-glucose, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), poly-glucose or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyoxypropylene/ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the MAGE-A4 tumor specific TCR or antigen binding domain thereof can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function to be improved of the MAGE-A4 tumor-specific TCR or antigen binding domain thereof, whether the MAGE-A4 tumor-specific TCR or antigen binding domain derivative thereof will be used in a defined condition therapy, and the like.

In some embodiments, conjugates of MAGE-A4 tumor specific TCRs or antigen binding domains thereof with non-protein moieties that can be selectively heated by exposure to radiation are provided. In some embodiments, the non-protein moiety is a carbon nanotube (Kam et al, proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may have any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein fraction to a temperature that kills cells proximal to the MAGE-A4 tumor specific TCR-non-protein fraction.

V. pharmaceutical composition

Also provided herein is a composition (e.g., a pharmaceutical composition, also referred to herein as a formulation) comprising a MAGE-A4 tumor-specific TCR or antigen binding domain thereof according to any of the embodiments described herein, a nucleic acid encoding a MAGE-A4 tumor-specific TCR or antigen binding domain thereof according to any of the embodiments described herein, or a MAGE-A4 tumor-specific TCR effector cell according to any of the embodiments described herein. In some embodiments, the composition is a MAGE-A4 tumor-specific TCR effector cell composition (e.g., a pharmaceutical composition) comprising effector cells (e.g., T cells) that present on the surface a MAGE-A4 tumor-specific TCR or antigen binding domain thereof according to any of the MAGE-A4 tumor-specific TCRs or antigen binding domains thereof described herein. In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition is a pharmaceutical composition. The composition may comprise a homogeneous cell population comprising MAGE-A4 tumor-specific TCR effector cells of the same cell type and expressing the same MAGE-A4 tumor-specific TCR or antigen binding domain thereof, or a heterogeneous cell population comprising a plurality of populations comprising MAGE-A4 tumor-specific TCR effector cells of different cell types and/or expressing different MAGE-A4 tumor-specific TCRs or antigen binding domains thereof. The composition may further comprise cells that are not MAGE-A4 tumor specific TCR effector cells. Thus, in some embodiments, a MAGE-A4 tumor-specific TCR effector cell composition is provided comprising a homogeneous population of MAGE-A4 tumor-specific TCR effector cells (e.g., MAGE-A4 tumor-specific TCRT cells) of the same cell type and expressing the same MAGE-A4 tumor-specific TCR. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, the MAGE-A4 tumor specific TCR effector 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, the MAGE-A4 tumor specific TCR effector cell composition is a pharmaceutical composition.

In some embodiments, a MAGE-A4 tumor-specific TCR effector cell composition is provided comprising a heterogeneous population of cells comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations comprising different cell types and/or MAGE-A4 tumor-specific TCR effector cells expressing different MAGE-A4 tumor-specific TCRs or antigen binding domains thereof. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population has a cell type selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, all MAGE-A4 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A4 tumor-specific TCR effector cells are cytotoxic T cells). In some embodiments, at least one MAGE-A4 tumor-specific TCR effector cell population has a different cell type than other MAGE-A4 tumor-specific TCR effector cell populations (e.g., one MAGE-A4 tumor-specific TCR effector cell population consists of cytotoxic T cells and the other MAGE-A4 tumor-specific TCR effector cell population consists of natural killer T cells). In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses the same MAGE-A4 tumor-specific TCR or antigen binding domain thereof. In some embodiments, at least one of the MAGE-A4 tumor-specific TCR effector cell populations expresses a MAGE-A4 tumor-specific TCR, or antigen binding domain thereof, that is different from the other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that is different from other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses MAGE-A4 tumor-specific TCRs, or antigen binding domains thereof, that specifically bind to the same target antigen. In some embodiments, at least one MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen that is different from other MAGE-A4 tumor-specific TCR effector cell populations (e.g., one MAGE-A4 tumor-specific TCR effector cell population specifically binds to the pMHC complex and other MAGE-A4 tumor-specific TCR effector cell populations specifically bind to a cell surface receptor). In some embodiments, wherein at least one population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a different target antigen, each population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a target antigen associated with the same disease or disorder (e.g., each of the target antigens is associated with cancer, such as breast cancer). In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition is a pharmaceutical composition.

Thus, in some embodiments, there is provided a MAGE-A4 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein all MAGE-A4 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A4 tumor-specific TCR effector cells are cytotoxic T cells), and wherein each MAGE-A4 tumor-specific TCR effector cell population expresses a different MAGE-A4 tumor-specific TCR or antigen binding domain thereof than the other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, the MAGE-A4 tumor specific TCR effector 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, each MAGE-A4 tumor-specific TCR effector cell population expresses MAGE-A4 tumor-specific TCRs, or antigen binding domains thereof, that specifically bind to the same target antigen. In some embodiments, at least one MAGE-A4 tumor-specific TCR effector cell population expresses a target antigen MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a different than other MAGE-A4 tumor-specific TCR effector cell populations (e.g., one MAGE-A4 tumor-specific TCR effector cell population specifically binds to the pMHC complex and other MAGE-A4 tumor-specific TCR effector cell populations specifically bind to a cell surface receptor). In some embodiments, wherein at least one population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a different target antigen, each population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a target antigen associated with the same disease or disorder (e.g., each of the target antigens is associated with cancer, such as breast cancer). In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition is a pharmaceutical composition.

In some embodiments, a composition is provided comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein at least one MAGE-A4 tumor-specific TCR effector cell population has a different cell type than the other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, all MAGE-A4 tumor specific TCR effector cell populations are of different cell types. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population has a cell type selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses the same MAGE-A4 tumor-specific TCR or antigen binding domain thereof. In some embodiments, at least one of the MAGE-A4 tumor-specific TCR effector cell populations expresses a MAGE-A4 tumor-specific TCR, or antigen binding domain thereof, that is different from the other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that is different from other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses MAGE-A4 tumor-specific TCRs, or antigen binding domains thereof, that specifically bind to the same target antigen. In some embodiments, at least one MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen that is different from other MAGE-A4 tumor-specific TCR effector cell populations (e.g., one MAGE-A4 tumor-specific TCR effector cell population specifically binds to the pMHC complex and other MAGE-A4 tumor-specific TCR effector cell populations specifically bind to a cell surface receptor). In some embodiments, wherein at least one population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a different target antigen, each population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or antigen-binding domain thereof, that specifically binds to a target antigen associated with the same disease or disorder (e.g., each of the target antigens is associated with cancer, such as breast cancer). In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition is a pharmaceutical composition.

At various points during the preparation of the composition, it may be desirable or desirable to cryopreserve the cells. The term "freezing (frozen/freezing)" and "cryopreservation (cryopreserved/cryopreserving)" are used interchangeably. Freezing includes freeze drying.

As will be appreciated by those skilled in the art, frozen cells can be destructive (see Mazur, P., 1977, cryobiology 14:251-272), but there are a number of procedures available for preventing such damage. For example, damage may be avoided by (a) using cryoprotectants, (b) controlling the rate of freezing, and/or (c) storing at a temperature low enough to minimize degradation response. Exemplary cryoprotectants include Dimethylsulfoxide (DMSO) (Lovelock and Bishop, 1959, nature 183:1394-1395; ashwood-Smith, 1961, nature 190:1204-1205), glycerol, polyvinylpyrrolidone (Rinfret, 1960, ann.N.Y. Acad.Sci.85: 576), polyethylene glycol (Sloviter and ravidin, 1962, nature 196:548), albumin, polydextrose, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al 1962, fed. Proc.21:157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al 1960, J.appl. Physiol.15:520), amino acids (PHAN THE TRAN and Bender 1960, exp. CellRes.20:651), methanol, acetamido, monoacetate (Lovelock, 1954, biochem. J.56:265) and inorganic salts (PHAN THE TRAN and Bender,1960, proc. Soc. Exp. Med. 104:388;PhanThe Tran and Bender 1961, radiobiology, 84, llbery, butterworth, london, p 59). In particular embodiments, DMSO may be used. The addition of plasma (e.g., to a concentration of 20-25%) may enhance the protective effect of DMSO. After DMSO addition, the cells may be kept at 0 ℃ until frozen, as 1% DMSO concentration may be toxic at temperatures above 4 ℃.

In cryopreservation of cells, slow controlled cooling rates may be critical and different cryoprotectants (Rapatz et al, 1968, cryobiology (1): 18-25) and different cell types have different optimal cooling rates (see, e.g., rowe and Rinfret,1962,Blood 20:636;Rowe,5 1966,Cryo biology3 (1): 12-18; lewis et al, 1967, transfusions 7 (1): 17-32; and Mazur,1970, science168: 939-949) for the effect of cooling rates on the viability of stem cells and their transplantation potential). The heat of the melting phase, where the water turns into ice, should be minimal. The cooling procedure may be performed by using, for example, a programmable freezer or a methanol bath procedure. The programmable freezing apparatus allows for determination of optimal cooling rates and facilitates standard reproducible cooling.

In particular embodiments, DMSO-treated cells can be pre-cooled on ice and transferred to trays containing chilled methanol, which in turn are placed in a mechanical refrigerator (e.g., harris or Revco) at-80 ℃. Thermocouple measurements of the methanol bath and sample indicated a cooling rate of 1 ℃ to 3 ℃/min may be preferred. After at least two hours, the sample may reach a temperature of-80 ℃ and may be placed directly in liquid nitrogen (-196 ℃). After bottom freezing, the cells can be rapidly transferred to long-term cryogenic storage vessels.

In a preferred embodiment, the sample may be stored cryogenically in liquid nitrogen (-196 ℃) or in vapor (-1 ℃). The availability of high efficiency liquid nitrogen refrigerators facilitates such storage. Other considerations and procedures for handling, cryopreservation and long term storage of cells can be found in the following exemplary references: U.S. Pat. Nos. 4,199,022, 3,753,357, 4,559,298, ; Gorin,1986,Clinics In Haematology 15(1):19-48Bone-Marrow Conservation,Culture and Transplantation, Proceedings of a Panel, Moscow,1968, 22-26 days, international Atomic ENERGY AGENCY, vienna, pages 107-186; livesey and Linner, 1987, nature 327:255; linner et al, 1986, J.Histochem. Cytochem.34 (9): 1123-1135; simione, 1992, J.Parenter. Sci. Technol.46 (6): 226-32.

After cryopreservation, the frozen cells may be thawed for use according to methods known to those skilled in the art. Frozen cells are preferably thawed rapidly and frozen immediately after thawing. In certain embodiments, vials containing frozen cells may be immersed in a warm water bath until their neck; gentle rotation will ensure that the cell suspension mixes with the thawing and increase the heat transfer from the warm water to the internal ice cubes. Once the ice is completely melted, the vials are placed on the ice immediately.

In certain embodiments, methods of preventing cell aggregation may be used during thawing. An exemplary method includes: deoxyribonuclease (Spitzer et al 113, 1980, cancer 45:3075-3085), low molecular weight polydextrose, citrate, hydroxyethyl starch (Stiff et al, 1983, cryobiology 20:17-24) and the like are added before and/or after freezing. As will be appreciated by those skilled in the art, if cryoprotectants toxic to humans are used, they should be removed prior to therapeutic use. DMSO is not severely toxic.

Exemplary vehicle and cell administration modes are described in U.S. patent publication No. 2010/0183564, pages 14-15. Other pharmaceutical carriers are described in Remington THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, division d.david b.troy, lippicottWilliams & Wilkins (2005).

In certain embodiments, cells can be collected from the culture medium and washed and concentrated in a carrier to a therapeutically effective amount. Exemplary carriers include saline, buffered saline, normal saline, water, hanks 'solution, ringer's solution (Ringer's solution)、Nonnosol-R(Abbott Labs)、Plasma-LyteA(R)(BaxterLaboratories,Inc.,MortonGrove,IL)、 glycerin, ethanol, and combinations thereof.

In particular embodiments, the carrier may be supplemented with Human Serum Albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, the infusion vehicle comprises buffered saline containing 5% has or dextrose. Other isotonic agents include polyhydroxy sugar alcohols including triols or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol.

The carrier may include buffers such as citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer, and/or trimethylamine salt.

Stabilizers refer to a broad class of excipients that can range in function from an extender to an additive that helps prevent cells from adhering to the walls of the container. Typical stabilizers may include polyhydroxy sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol, and cyclic alcohols, such as inositol; PEG; an amino acid polymer; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides, such as raffinose; and polysaccharides such as polydextrose.

The composition may include a local anesthetic such as lidocaine (lidocaine) to reduce pain at the injection site, if desired or advantageous.

Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, octadecyl dimethyl benzyl ammonium chloride, benzalkonium halides, quaternary ammonium hexa-hydroxy chloride, alkyl parahydroxybenzoates (e.g., methyl or propyl parahydroxybenzoate), catechol, resorcinol, cyclohexanol, and 3-pentanol.

The therapeutically effective amount of cells within the composition can be greater than 10 ² cells, greater than 10 ³ cells, greater than 10 ⁴ cells, greater than 10 ⁵ cells, greater than 10 ⁶ cells, greater than 10 ⁷ cells, greater than 10 ⁸ cells, greater than 10 ⁹ cells, greater than 10 ¹⁰ cells, or greater than 10 ¹¹ cells. In the compositions and formulations disclosed herein, the volume of the cells is typically one liter or less, 500ml or less, 250ml or less, or 100ml or less. Thus, the cell density administered is typically greater than 10 ⁴ cells/ml, 10 ⁷ cells/ml, or 10 ⁸ cells/ml.

Also provided herein are MAGE-A4 tumor-specific TCR nucleic acid compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any of the nucleic acids encoding MAGE-A4 tumor-specific TCRs described herein or antigen binding domains thereof. In some embodiments, the MAGE-A4 tumor specific TCR nucleic acid composition is a pharmaceutical composition. In some embodiments, the MAGE-A4 tumor specific TCR nucleic acid composition further comprises any one of: isotonic agents, excipients, diluents, thickeners, stabilizers, buffers and/or preservatives; and/or an aqueous vehicle, such as purified water, aqueous sugar solution, buffer solution, physiological saline, aqueous polymer solution, or water free of ribonuclease. The amount of such additives and aqueous vehicles to be added may be appropriately selected depending on the form of use of the MAGE-A4 tumor specific TCR nucleic acid composition.

The compositions and formulations disclosed herein may be prepared for administration by, for example, injection, infusion, perfusion, or lavage. The compositions and formulations may further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodular, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intracapsular, and/or subcutaneous injection.

Formulations for in vivo administration must be sterile. This requirement is easily achieved by filtration, for example through sterile filtration membranes. Methods of treatment using a MAGE-A4 tumor-specific TCR or antigen-binding domain thereof the MAGE-A4 tumor-specific TCR or antigen-binding domain thereof and/or compositions of the application can be administered to an individual (e.g., a mammal, such as a human) to treat diseases and/or disorders associated with expression of a Target Antigen (TA) (also referred to herein as "target antigen positive" or "TA positive" diseases or disorders), including, for example, cancer. Thus in some embodiments, the application provides a method for treating a target antigen positive disease (e.g., cancer) in an individual comprising administering to the individual an effective amount of a composition (e.g., a pharmaceutical composition) comprising, for example, any of the MAGE-A4 tumor specific TCRs described herein or antigen binding domains thereof.

In some embodiments, the composition further comprises a cell (e.g., an effector cell) associated with the MAGE-A4 tumor specific TCR or antigen binding domain thereof. In some embodiments, the cancer is selected from, for example, the group consisting of: adrenal cortex cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, liver cancer, head and neck cancer, renal cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer, and thyroid cancer.

VI therapeutic methods

In some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in an individual in need thereof is provided, comprising administering to the individual an effective amount of a composition comprising a MAGE-A4 tumor-specific TCR, or an antigen-binding domain thereof, according to any of the herein, or a nucleic acid encoding a MAGE-A4 tumor-specific TCR, or an antigen-binding domain thereof, according to any of the herein, or an effector cell (e.g., T cell or natural killer cell) presenting a MAGE-A4 tumor-specific TCR, or an antigen-binding domain thereof, on a surface (e.g., a MAGE-A4 tumor-specific TCR, or an antigen-binding domain thereof). In some embodiments, the above-described composition comprises a) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO. 9 and b) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO. 12; and/or c) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO. 23 and d) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO. 26; and/or e) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO. 37 and f) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO. 40. In some embodiments, effector cells expressing a MAGE-A4 tumor specific TCR or antigen binding domain thereof further comprise a P2A nucleic acid sequence encoding SEQ ID NOs: 13, 27, 41. In some embodiments, an effector cell (e.g., T cell) is provided that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on a surface comprising a nucleic acid sequence encoding the amino acid sequence of one or more of SEQ ID NOs 14, 28, 42.

In some embodiments, the above composition comprises an isolated MAGE-A4 TCR or antigen binding domain thereof comprising a) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO. 9 and b) a TCR beta chain comprising the amino acid sequence of SEQ ID NO. 12; and/or c) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO. 23 and d) a TCR beta chain comprising the amino acid sequence of SEQ ID NO. 26; and/or e) a TCR alpha chain comprising the amino acid sequence of SEQ ID NO. 37 and f) a TCR beta chain comprising the amino acid sequence of SEQ ID NO. 40. In some embodiments, the MAGE-A4 tumor specific TCR or antigen binding domain thereof further comprises the P2A sequence of SEQ ID NOs: 13, 27, 41. In some embodiments, a MAGE-A4 tumor specific TCR or antigen binding domain thereof is provided comprising the amino acid sequence of one or more of SEQ ID NOs 14, 28, 42.

In some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in a subject in need thereof is provided, comprising administering to the subject an effective amount of a composition comprising an effector cell (e.g., a T cell or natural killer cell) that presents a MAGE-A4 tumor-specific TCR or antigen binding domain thereof (e.g., a MAGE-A4 tumor-specific TCR or antigen binding domain thereof) on a surface, wherein the target antigen of the effector cell (e.g., T cell) of the MAGE-A4 tumor-specific TCR is a MAGE-A4 peptide/MHC complex. In some embodiments, the MHC protein in the MAGE-A4 peptide/MHC complex is an MHC-class I protein. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, the effector cell is a γδ T cell. In some embodiments, the effector cell is an αβ T cell. In some embodiments, the effector 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, the target antigen of an effector cell (e.g., T cell) that expresses a MAGE-A4 tumor specific TCR or antigen binding domain thereof on the surface is a MAGE-A4 _146-154 peptide/mhc i a 11:01 complex. The invention also encompasses a method of treating a target antigen-related disease in an individual in need thereof comprising administering to the individual a composition comprising a plurality of effector cells expressing different MAGE-A4 tumor-specific TCRs or antigen binding domains thereof. Thus, in some embodiments, the composition is a heterogeneous MAGE-A4 tumor-specific TCR effector cell composition as described herein, according to any of the methods of treating a target antigen-related disease in an individual described herein. For example, in some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in an individual in need thereof is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A4 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein all MAGE-A4 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A4 tumor-specific TCR effector cells are cytotoxic T cells), wherein each MAGE-A4 tumor-specific TCR effector cell population expresses a different MAGE-A4 tumor-specific TCR than the other MAGE-A4 tumor-specific TCR effector cell populations, and wherein at least one MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR effector cell that specifically binds to the target antigen. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, the MAGE-A4 tumor specific TCR effector 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, each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen. In some embodiments, at least one population of MAGE-A4 tumor-specific TCR effector cells expresses MAGE-A4 tumor-specific TCRs or antigen binding domains thereof that specifically bind to different target antigens. In some embodiments, wherein at least one population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or an antigen binding domain thereof, that specifically binds to a different target antigen, each of which is associated with a target antigen-related disease.

In some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in an individual in need thereof is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A4 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein at least one MAGE-A4 tumor-specific TCR effector cell population has a different cell type than the other MAGE-A4 tumor-specific TCR effector cell populations, and wherein at least one MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to the target antigen. In some embodiments, all MAGE-A4 tumor specific TCR effector cell populations are of different cell types. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population has a cell type selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses the same MAGE-A4 tumor-specific TCR or antigen binding domain thereof. In some embodiments, at least one of the MAGE-A4 tumor-specific TCR effector cell populations expresses a MAGE-A4 tumor-specific TCR, or antigen binding domain thereof, that is different from the other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that is different from other MAGE-A4 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen. In some embodiments, at least one population of MAGE-A4 tumor-specific TCR effector cells expresses MAGE-A4 tumor-specific TCRs or antigen binding domains thereof that specifically bind to different target antigens. In some embodiments, wherein at least one population of MAGE-A4 tumor-specific TCR effector cells expresses a MAGE-A4 tumor-specific TCR, or an antigen binding domain thereof, that specifically binds to a different target antigen, each of which is associated with a target antigen-related disease.

In some embodiments, a method of treating a disease associated with a plurality of target antigens in an individual in need thereof is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A4 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein all MAGE-A4 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A4 tumor-specific TCR effector cells are cytotoxic T cells), wherein each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that is different from the other MAGE-A4 tumor-specific TCR effector cell populations, and wherein at least one MAGE-A4 tumor-effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to a target antigen for each of the plurality of target antigens. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, the MAGE-A4 tumor specific TCR effector 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, a method of treating a disease associated with a plurality of target antigens in an individual in need thereof is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A4 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A4 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein at least one MAGE-A4 tumor-specific TCR effector cell population has a different cell type than the other MAGE-A4 tumor-specific TCR effector cell populations, and wherein for each target antigen of the plurality of target antigens, at least one MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that specifically binds to the target antigen. In some embodiments, all MAGE-A4 tumor specific TCR effector cell populations are of different cell types. In some embodiments, the MAGE-A4 tumor specific TCR effector cell is a T cell. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population has a cell type selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A4 tumor-specific TCR effector cell population expresses a MAGE-A4 tumor-specific TCR or antigen binding domain thereof that is different from other MAGE-A4 tumor-specific TCR effector cell populations.

In some embodiments, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the subject is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, a laboratory animal, or the like. In some embodiments, the individual is less than about 60 years old (including, for example, any of ages less than about 50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, the individual is older than about 60 years (including, for example, any of ages older than about 70, 80, 90, or 100). In some embodiments, the individual is diagnosed with or is environmentally or genetically predisposed to one or more of the diseases or disorders described herein (e.g., cancer). In some embodiments, the individual has one or more risk factors associated with one or more of the diseases or conditions described herein.

In some embodiments, the MAGE-A4 tumor specific TCR effector cell compositions of the invention are administered in combination with a second, third, or fourth agent (including, for example, an anti-neoplastic agent, a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder involving expression of a target antigen. In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition is administered in combination with a cytokine (e.g., IL-2). In some embodiments, the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, is administered in combination with an agent that increases MHC protein expression and/or enhances surface presentation of the peptide by the MHC protein. In some embodiments, the agents include, for example, IFN receptor agonists, hsp90 inhibitors, p53 expression enhancers, and chemotherapeutic agents. In some embodiments, the agent is an IFN receptor agonist, including for example IFN gamma, IFN beta and IFN alpha. In some embodiments, the agent is an Hsp90 inhibitor, including, for example, tamsulosin (TANESPIMYCIN; 17-AAG), azithromycin (ALVESPIMYCIN; 17-DMAG), rithromycin (RETASPIMYCIN; IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, debio0932 (CUDC-305), PU-H71, and Li Tepi (Ganetespib; STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248, and XL888. In some embodiments, the agent is a p53 expression enhancer, including, for example, 5-fluorouracil and nutlin-3. In some embodiments, the agent is a chemotherapeutic agent, including, for example, topotecan (topotecan), etoposide (etoposide), cisplatin (cispratin), paclitaxel (paclitaxel), and vinblastine (vinblastine). In some embodiments, a method of treating a target antigen-positive disease in a subject in need thereof is provided, comprising administering to the subject a MAGE-A4 tumor-specific TCR effector cell composition according to any of the embodiments described herein, and a cytokine (e.g., IL-2). In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition and cytokine are administered simultaneously. In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition and cytokine are administered sequentially.

In some embodiments, a method of treating a target antigen-positive disease in an individual in need thereof is provided, wherein cells expressing the target antigen are not normally presented or present at relatively low levels on their surfaces a complex comprising the target antigen and an mhc class of proteins, the method comprising administering to the individual a MAGE-A4 tumor-specific TCR effector cell composition according to any of the embodiments described herein and an agent that increases expression of the mhc class of proteins and/or enhances surface presentation of the target antigen by the mhc class of proteins. In some embodiments, the agents include, for example, IFN receptor agonists, hsp90 inhibitors, p53 expression enhancers, and chemotherapeutic agents. In some embodiments, the agent is an IFN receptor agonist, including for example IFN gamma, IFN beta and IFN alpha. In some embodiments, the agent is an Hsp90 inhibitor, including, for example, tamsulosin (17-AAG), azithromycin (17-DMAG), rithromycin (IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, debio0932 (CUDC-305), PU-H71, and Li Tepi (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248, and XL888. In some embodiments, the agent is a p53 expression enhancer, including, for example, 5-fluorouracil and nutlin-3. In some embodiments, the agent is a chemotherapeutic agent, including, for example, topotecan, etoposide, cisplatin, paclitaxel, and vinblastine. In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition and the agent are administered simultaneously. In some embodiments, the MAGE-A4 tumor specific TCR effector cell composition and the agent are administered sequentially.

In some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in an individual in need thereof is provided, comprising administering to the individual an effective amount of a composition comprising a nucleic acid encoding a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, according to any of the embodiments described herein. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety.

Cancer treatment can be assessed by, for example, tumor regression, tumor weight or size shrinkage, percent cancer cell death, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression, and/or activity. Methods for determining efficacy of a therapy may be used, including measuring response, for example, via radiological imaging.

In some embodiments, the efficacy of treatment is measured as percent tumor growth inhibition (TGI%), calculated using formula 100- (T/C x 100), where T is the average relative tumor volume of the treated tumor and C is the average relative tumor volume of the untreated tumor. In some embodiments, the TGI% is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%.

In some embodiments, the therapeutic efficacy is measured as percent tumor growth inhibition (TGI%), calculated using formula 100- (T/C x 100), where T is the number of treated target cells and C is the number of untreated target cells. In some embodiments, the TGI% is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%.

In some embodiments, according to any of the methods of treating a target antigen-related disease in an individual described herein, further in combination with an immunosuppressant, a therapeutic antibody, chemotherapy, radiation therapy, surgery, or any combination thereof.

Disease of the human body

In some embodiments, MAGE-A4 tumor specific TCR effector cells can be useful for treating cancers associated with a target antigen. Cancers that may be treated using any of the methods described herein include non-vascularized, or tumors that have not been substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (e.g., hematological tumors such as leukemia and lymphoma) or may comprise a solid tumor. Types of cancers to be treated by the MAGE-A4 tumor-specific TCR effector cells of the invention include, but are not limited to, carcinoma, blastoma and sarcoma, as well as certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant diseases, such as sarcoma, carcinoma and melanoma. Adult tumors/cancers and pediatric tumors/cancers are also included. Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include leukemias, including acute leukemias (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia and myeloblastosis, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-Hodgkin's lymphomas (refractory and advanced forms), multiple myelomas, plasmacytomas, waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia), heavy chain diseases, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. Solid tumors are abnormal tissue masses that do not typically contain cysts or liquid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named by the cell type from which they are formed (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors (e.g., sarcomas and carcinomas) include adrenocortical, cholangiocarcinomas, fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteosarcomas and other sarcomas, synoviomas, mesotheliomas, ewing ' stumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, gastric cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullary thyroid carcinoma and papillary thyroid carcinoma), pheochromocytoma sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, wilms ' tumor (Wilms ' tumor), cervical cancer (e.g., cervical cancer and pre-invasive cervical dysplasia), colorectal cancer, anal canal cancer or anal rectal cancer, vaginal cancer, vulvar cancer (e.g., squamous cell carcinoma, intraepithelial cancer, adenocarcinoma, and fibrosarcoma), penile cancer, oropharyngeal cancer, esophageal cancer, head cancer (e.g., squamous cell carcinoma), neck cancer (e.g., squamous cell carcinoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, leydig cell tumor (Leydigcelltumor), fibroma, fibroadenoma, adenomatoid tumor, and lipoma), bladder cancer, renal cancer, melanoma, uterine cancer (e.g., endometrial cancer), urothelial cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral cancer, and bladder cancer), and CNS tumors (e.g., gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastoma (also known as polymorphic glioblastoma) astrocytomas, CNS lymphomas, blastomas, neuroblastomas, neurosheath tumors, craniopharyngeal tube tumors, ependymomas, pineal tumors, angioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

Cancer treatment may be assessed, for example, by tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression, and/or activity. Methods for determining efficacy of a therapy may be used, including measuring response, for example, via radiological imaging.

Product and kit

In some embodiments of the invention, an article of manufacture is provided that contains a material suitable for treating a target antigen-positive disease, such as cancer (e.g., adrenocortical, bladder, breast, cervical, cholangiocarcinoma, colorectal, esophageal, glioblastoma, glioma, hepatocellular, head and neck, renal, lung, melanoma, mesothelioma, multiple myeloma, pancreatic, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian, prostate, sarcoma, gastric, uterine, or thyroid cancer). The article of manufacture may comprise a container, a label on or accompanying the container, or a pharmaceutical instruction. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of various materials, such as glass or plastic. In general, the container contains a composition effective for treating the diseases or conditions described herein, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an effector cell that presents on the surface a MAGE-A4 tumor specific TCR of the invention or an antigen binding domain thereof. The label or pharmaceutical instructions indicate that the composition is used to treat a particular condition. The labeling or pharmaceutical instructions will further comprise instructions for administering to the patient a MAGE-A4 tumor specific TCR effector cell composition. Articles of manufacture and kits comprising the combination therapies described herein are also contemplated.

The pharmaceutical instructions refer to instructions that are typically included in commercial therapeutic product packages that contain instructions for indications, uses, dosages, administration, contraindications and/or warnings regarding the use of such therapeutic products. In some embodiments, the pharmaceutical instructions indicate that the composition is used to treat a target antigen positive cancer (e.g., adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, hepatocellular carcinoma, head and neck cancer, renal cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer, or thyroid cancer). Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other substances, including other buffers, diluents, filters, needles and syringes, as desired from a commercial and user standpoint.

Kits suitable for various purposes, such as for treating a target antigen positive disease or disorder described herein, optionally in combination with an article of manufacture, are also provided. Kits of the invention include one or more containers comprising a MAGE-A4 tumor specific TCR effector cell composition (or unit dosage form and/or article of manufacture), and in some embodiments, further comprising another agent (as described herein) and/or instructions for use according to any of the methods described herein. The kit may further comprise instructions for the selection of individuals suitable for treatment. The instructions provided in the kits of the invention are written instructions that are typically on labels or pharmaceutical instructions (e.g., paper sheets included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetized or optical storage disc) are also acceptable.

For example, in some embodiments, the kit comprises a composition comprising effector cells that present a MAGE-A4 tumor specific TCR on a surface. In some embodiments, the kit comprises a) a composition comprising effector cells that present a MAGE-A4 tumor specific TCR or antigen binding domain thereof on a surface, and b) an effective amount of at least one additional agent, wherein the additional agent increases expression of an MHC protein and/or enhances surface presentation of the MHC protein (e.g., ifnγ, ifnβ, ifnα, or Hsp90 inhibitor) to the peptide. In some embodiments, the kit comprises a) a composition comprising effector cells that present on the surface a MAGE-A4 tumor specific TCR or antigen binding domain thereof, and b) administering to the individual

Instructions for MAGE-A4 tumor specific TCR effector cell compositions to treat target cell positive diseases (e.g., cancer). In some embodiments, the kit comprises a) a composition comprising effector cells that present a MAGE-A4 tumor-specific TCR or antigen binding domain thereof on a surface, b) an effective amount of at least one additional agent, wherein the additional agent increases expression of MHC proteins and/or enhances surface presentation of peptides by MHC proteins (e.g., ifnγ, ifnβ, ifnα, or Hsp90 inhibitors), and c) instructions for administering the MAGE-A4 tumor-specific TCR effector cell composition and additional agent to an individual to treat a target cell positive disease (e.g., cancer). The MAGE-A4 tumor specific TCR effector cell composition and other agents can be present in separate containers or in a single container. For example, the kit may comprise one unique composition or two or more compositions, wherein one composition comprises MAGE-A4 tumor specific TCR effector cells and the other composition comprises another agent.

In some embodiments, the kit comprises a) a composition comprising a MAGE-A4 tumor-specific TCR or antigen-binding domain thereof, and b) instructions relating to combining the MAGE-A4 tumor-specific TCR or antigen-binding domain thereof with an effector cell (e.g., an effector cell derived from an individual, such as a T cell or a natural killer cell) to form a composition comprising an effector cell presenting the MAGE-A4 tumor-specific TCR or antigen-binding domain thereof on a surface, and administering the MAGE-A4 tumor-specific TCR effector cell composition to the individual to treat a target antigen-positive disease (e.g., cancer). In some embodiments, the kit comprises a) a composition comprising a MAGE-A4 tumor specific TCR, or an antigen binding domain thereof, and b) an effector cell (e.g., a cytotoxic cell). In some embodiments, the kit comprises a) a composition comprising a MAGE-A4 tumor-specific TCR or antigen binding domain thereof, b) an effector cell (e.g., a cytotoxic cell), and c) instructions for combining the MAGE-A4 tumor-specific TCR or antigen binding domain thereof with the effector cell to form a composition comprising an effector cell that presents the MAGE-A4 tumor-specific TCR or antigen binding domain thereof on a surface, and administering the MAGE-A4 tumor-specific TCR effector cell composition to a subject to treat a target antigen-positive disease (e.g., cancer).

In some embodiments, the kit comprises one nucleic acid (or a set of nucleic acids) encoding a MAGE-A4 tumor specific TCR or antigen binding domain thereof. In some embodiments, the kit comprises a) a nucleic acid (or a set of nucleic acids) encoding a MAGE-A4 tumor specific TCR or antigen binding domain thereof, and b) a host cell (e.g., an effector cell) for expressing the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises a) a nucleic acid (or a set of nucleic acids) encoding a MAGE-A4 tumor-specific TCR or antigen binding domain thereof, and b) instructions for i) expressing the MAGE-A4 tumor-specific TCR or antigen binding domain thereof in a host cell (e.g., an effector cell, such as a T cell), ii) preparing a composition comprising a host cell that expresses the MAGE-A4 tumor-specific TCR or antigen binding domain thereof, and iii) administering to the subject the composition comprising a host cell that expresses the MAGE-A4 tumor-specific TCR or antigen binding domain thereof, to treat the target antigen-positive disease (e.g., cancer). In some embodiments, the host cell is derived from an individual. In some embodiments, the kit comprises a) a nucleic acid (or a set of nucleic acids) encoding a MAGE-A4 tumor-specific TCR or antigen binding domain thereof, b) a host cell (e.g., an effector cell) for expressing the nucleic acid (or the set of nucleic acids), and c) instructions for i) expressing the MAGE-A4 tumor-specific TCR or antigen binding domain thereof in the host cell, ii) preparing a composition comprising a host cell expressing the MAGE-A4 tumor-specific TCR or antigen binding domain thereof, and iii) administering to the subject a composition comprising a host cell expressing the MAGE-A4 tumor-specific TCR or antigen binding domain thereof, to treat the target antigen-positive disease (e.g., cancer). The kit of the application is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, cans, plastic packages (e.g., sealed Mylar or plastic bags), and the like. The kit may optionally provide additional components such as buffers and instructional information. The present application thus also provides articles including vials (e.g., sealed vials), bottles, cans, plastic packages, and the like.

Instructions relating to the use of MAGE-A4 tumor specific TCR effector cell compositions generally include information regarding the dosage, timing of administration, and route of administration of the desired treatment. The container may be a unit dose, a bulk (e.g., multi-dose package) or a sub-unit dose. For example, a kit can be provided containing a sufficient dose of a MAGE-A4 tumor specific TCR effector cell composition as disclosed herein for providing an effective treatment for an individual for a longer period of time, e.g., any one of one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more.

The kit may also include a plurality of unit doses of the MAGE-A4 tumor specific TCR, or antigen binding domain thereof, and pharmaceutical compositions and instructions for use, and packaged in amounts sufficient for storage and use in a pharmacy, such as a hospital pharmacy and pharmacy.

Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail with reference to the following non-limiting examples. The following embodiments further illustrate the invention but, of course, should not be construed as in any way limiting its scope. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the invention.

Detailed Description

Example 1 preparation of MAGE-A4 specific T cells

A MAGE-A4 antigen-specific T cell receptor ST18G02 was prepared for characterization and validation experiments. The HLA and epitope of the tumor antigen specific T cell receptor are shown in FIG. 1A. For each tumor antigen-specific T cell receptor, preparing a human (i.e., wild-type) TCR construct and a murine TCR construct corresponding thereto; codon optimization was performed for ST18G 02. The murine TCR construct has murine constant domains (mcα and mcβ1). Schematic of the wild-type, murine and murine codon-optimized constructs for tumor antigen specific T cell receptors are shown in fig. 1B.

Example 2 verification of MAGE-A4 specific T cell receptor ST18G02

This example aims at verifying T Cell Receptor (TCR) ST18G02 expression, polypeptide recognition, epitope recognition, HLA blocking and in vitro toxicity.

TCR expression and polypeptide recognition assays

The TCR expression and polypeptide recognition assay is shown in figure 2A.

TCR expression was assessed using FACS assays that detect stained TCR vβ chains. Briefly, TCR-transferred T cells were collected and washed by addition of 10 mL PBS (containing 2% fetal bovine serum), centrifuged at 350g for 5min, and the supernatant was completely aspirated. The cell pellet was resuspended and adjusted to about 2×10 ⁶ cells/mL by adding PBS (containing 2% fetal bovine serum). 100 μl/sample cell suspension was collected and used for TCR vβ chain surface staining and detected by FACS.

Recognition of MAGE-A4 by TCR transduced T cells was determined using MAGE-A4 _146-154 polypeptide. TCR transduced T cells were collected and washed by addition of 10mL PBS, centrifuged at 350g for 5 min, and the supernatant was completely aspirated. TCR-transduced T cells were then resuspended by addition of AIM-V medium (containing 10% fetal bovine serum) and adjusted to 1x 10 ⁶ cells/mL into 96-well plates. MAGE-A4 _146-154 polypeptide was mixed with tumor specific T cells, brix A (brefeldin A) (final concentration 3 μg/mL) was added to the wells and incubated for 4 hours. After 4 hours, cells were collected and used for intracellular IFN-. Gamma.and CD3 staining and detection by FACS. For ELISA detection of IFN-. Gamma.100. Mu.L/well of LCL (1X 10 ⁶ cells/mL) and 100. Mu.L/well of TCR transduced T cells (1X 10 ⁶ cells/mL) were mixed into 96-well plates and incubated in an incubator for 24 hours. After 24 hours, 175 μl of supernatant was collected per well in 96 discs and used for ELISA detection of IFN- γ using the IFN- γ ELISA HRP kit.

As shown in FIGS. 2A-2C, ST18G02 specifically recognizes the polypeptide MAGE-A4 _146-154.

LCL stimulation assay for validation of epitope recognition

LCL stimulation assays were performed as follows. First, LCL was loaded with peptide: LCLs were collected in 15 mL tubes, washed by addition of 10mL PBS, centrifuged at 350g for 5 min, and the supernatant was completely aspirated. The cell pellet was resuspended in medium (RPMI 1640 with 10% FBS) and adjusted to 1×10 ⁶ cells/mL. Tumor antigen peptides were added to LCL to a final concentration of 5 μg/mL and incubated in an incubator for 8 to 24 hours. The TCR-T cells were then stimulated with peptide-loaded LCLs as follows: the peptide-loaded LCL was collected and washed by addition of 10mL PBS, centrifuged at 350g for 5 min, and the supernatant was completely aspirated. LCL was resuspended and adjusted to 1X 10 ⁶ cells/mL by adding AIM-V medium (containing 10% fetal bovine serum). TCR transduced T cells were collected and washed by addition of 10mL PBS, centrifuged at 350g for 5 min, and the supernatant was completely aspirated. The TCR transduced T cells were then resuspended by addition of AIM-V medium (containing 10% fetal bovine serum) and adjusted to 1X 10 ⁶ cells/mL. 100 μl/well of LCL (1×10 ⁶ cells/mL) and 100 μl/well of TCR-transduced T cells (1×10 ⁶ cells/mL) were mixed into 96-well plates and incubated in an incubator for 24 hours. After 24 hours, 175 μl of supernatant was collected per well in 96 discs and used for ELISA detection of IFN- γ using the IFN- γ ELISA HRP kit.

HLA restriction assay

Cells with various HLA-A subtypes are loaded with MAGE-A4 _146-154 polypeptide to stimulate T cells transfected with T cell receptors in an HLA restriction assay. Washed by addition of 10mL PBS g, centrifuged at 350g for 5 min and the supernatant was completely aspirated. The cell pellet was resuspended to 1X 10 ⁶ cells/mL by addition of AIM-V medium (containing 10% fetal bovine serum). 100 μl/well of cell suspension was added to the 96-well plate, and HLA blocking antibodies were added (final concentration 50 μg/mL) to the appropriate wells and incubated in an incubator for 2 hours. TCR-transferred T cells were collected, washed by adding 10mL PBS, centrifuged at 350g for 5 min, and the supernatant was completely aspirated. Cell pellet was collected and adjusted to 1X 10 ⁶ cells/mL by adding AIM-V medium (containing 10% fetal bovine serum). 100 μl/well of TCR transduced cell suspension was added to the corresponding well, mixed with LCL, and cultured in an incubator for 24 hours. After 24 hours, 175 μl of supernatant was collected per well of 96 discs and finally measured using a human IFN- γ ELISA.

On day 1, high protein binding ELISA plates were used for antibody 1-D1K (IFN- γ ELISA HRP kit) coating (by addition of 50 μl/well) diluted to 2 μg/mL in PBS (pH 7.4) and incubated overnight at 4 ℃. On day 2, the discs were washed twice with PBS (200 μl/well). The discs were blocked by adding 200 μl/well of medium and incubated for 1 hour at Room Temperature (RT). Human IFN-gamma standard (IFN-gamma ELISA HRP kit) was prepared in 2 mL PBS (with 1% BSA) to a concentration of 0.5 μg/mL and placed at RT for 15 min, after which the tube was vortexed. Samples or standards of 50 μl/well were diluted in medium and incubated for 2 hours at RT. The sample and standard probes were tested in duplicate. The discs were washed five times with PBS containing 0.05% Tween 20. 50. Mu.L/well of antibody 7-B6-1-biotin (IFN-. Gamma.ELISA HRP kit) was added at 1. Mu.g/mL (in PBS), incubated for 1 hour at RT, and washed. 50 μl/well of streptavidin-HRP (IFN- γ ELISA HRP kit) diluted 1:1000 in PBS was added, incubated for 1 hour at RT, and washed. 100 μl/well of TMB substrate solution was added and incubated in the dark at RT for 15 to 30 minutes until the solution turned visible blue in the well. A 50 μl/well of stop solution was added to terminate the enzymatic reaction. The color of the solution changed from blue to yellow. The light intensity was measured with an ELISA reader at 450 nm.

As shown in FIG. 3, T cells transfected with ST18G02 recognized the MAGE-A4 _146-154 antigen peptide presented by HLA-A 11:01.

In vitro toxicity assay

T cells transfected with T cell receptor ST18G02 as effector cells at 2:1 was mixed with target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

As shown in FIGS. 4-5, T cells transfected with ST18G02 had significant killing effect on A549 target cells expressing HLA-A 11:01 and MAGE-A4 proteins, 53.79% of the target cells were killed. The untransfected T cells in the control group had no killing effect on the target cells.

Example 3 verification of MAGE-A4 specific T cell receptor ST18G02-AM12

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM12, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

The TCR expression and polypeptide recognition assay is shown in figure 2A. The specific experimental procedure was the same as in example 2.

As shown in FIG. 6, ST18G02-AM12 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM12 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 7-8 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM12 in vitro on NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM12 had a significant killing effect on NCI-H520 target cells carrying MAGE-A4 protein, 95.72% of the target cells were killed.

Example 4 verification of MAGE-A4 specific T cell receptor ST18G02-AM17

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM17, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 9, ST18G02-AM17 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM17 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 10-11 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM17 in vitro on NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM17 had a significant killing effect on NCI-H520 target cells carrying MAGE-A4 protein, 82.17% of the target cells were killed.

Example 5 verification of MAGE-A4 specific T cell receptor ST18G02-AM26

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM26, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 12, ST18G02-AM26 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM26 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 13-14 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM26 in vitro on NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM26 had a significant killing effect on NCI-H520 target cells carrying MAGE-A4 protein, 94.23% of the target cells were killed.

Example 6 verification of MAGE-A4 specific T cell receptor ST18G02-AM31

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM31, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 15, ST18G02-AM31 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM31 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 16-17 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM31 in vitro on NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM31 had a significant killing effect on NCI-H520 target cells carrying MAGE-A4 protein, and 93.00% of the target cells were killed.

Example 7 MAGE-A4 verification of the specific T cell receptor ST18G02-AM40

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM40, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 18, ST18G02-AM40 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM40 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 19-20 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM40 in vitro on NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM40 had a significant killing effect on NCI-H520 target cells carrying MAGE-A4 protein, 94.23% of the target cells were killed.

Example 8 verification of MAGE-A4 specific T cell receptor ST18G02-AM41

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM41, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 21, ST18G02-AM41 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM41 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 22-23 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM41 in vitro on A375 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM41 had significant killing effect on A375 target cells carrying MAGE-A4 protein, 83.55% of the target cells were killed.

Example 9 verification of MAGE-A4 specific T cell receptor ST18G02-AM43

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM43, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 24, ST18G02-AM43 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM43 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 25-26 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM43 in vitro on NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM43 had a significant killing effect on NCI-H520 target cells carrying MAGE-A4 protein, 83.12% of the target cells were killed.

Example 10 verification of MAGE-A4 specific T cell receptor ST18G02-AM54

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM54, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 27, ST18G02-AM54 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM54 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were A375 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 28-29 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM54 in vitro on A375 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM54 had significant killing effect on A375 target cells carrying MAGE-A4 protein, 92.97% of the target cells were killed.

Example 11 verification of MAGE-A4 specific T cell receptor ST18G02-AM57

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM57, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 30, ST18G02-AM57 may specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM57 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were NCI-H520 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 31-32 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM57 in vitro on A375 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM57 had significant killing effect on A375 target cells carrying MAGE-A4 protein, 86.00% of the target cells were killed.

Example 12 verification of MAGE-A4 specific T cell receptor ST18G02-AM60

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM60, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 33, ST18G02-AM60 can specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM60 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were A375 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 34-35 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM60 in vitro on A375 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM60 had significant killing effect on A375 target cells carrying MAGE-A4 protein, 88.48% of the target cells were killed.

Example 13 verification of MAGE-A4 specific T cell receptor ST18G02-AM81

This example aims at verifying the expression of T Cell Receptor (TCR) ST18G02-AM81, polypeptide recognition, epitope recognition and in vitro toxicity.

TCR expression and polypeptide recognition assays

As shown in FIG. 36, ST18G02-AM81 can specifically recognize the polypeptide MAGE-A4 _146-154.

In vitro toxicity assay

T cells transfected with T cell receptors ST18G02 and ST18G02-AM81 were used as effector cells at 2:1 was mixed with the target cells as an experimental group. The target cells used in this experiment were H1299 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells not transfected with T cell receptor were mixed with target cells in the same ratio as a control group. The killing power of effector cells on target cells was measured continuously for 24 hours in the experimental group and the control group.

FIGS. 37-38 show cytotoxicity of T cells expressing the MAGE-A4 antigen specific T cell receptor ST18G02 and ST18G02-AM81 in vitro on H1299 cells expressing HLA-A 11:01 and MAGE-A4 proteins. T cells transfected with ST18G02-AM81 had a significant killing effect on H1299 target cells carrying MAGE-A4 protein, 64.06% of the target cells were killed.

CDR sequences of TCRs of the invention

The sequence related to the invention is as follows

Sequence number	Annotating	Sequence(s)
			1	ST18G02α-CDR1	NTAFDY（SEQ ID NO.1）
2	ST18G02α-CDR2	IRPDVSE（SEQ ID NO.2）
			3	ST18G02α-CDR3	CAAPGEDSNYQLIW（SEQ ID NO.3）
4	ST18G02β-CDR1	MNHEY（SEQ ID NO.4）
			5	ST18G02β-CDR2	SMNVEV（SEQ ID NO.5）
6	ST18G02β-CDR3	CASSLSVGDTQYF（SEQ ID NO.6）
			7	ST18G02Vα-Jα	MDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPD（SEQ ID NO.7）
8	ST18G02Cα	IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.8）
			9	ST18G02 alpha chain	MDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.9）
10	ST18G02Vβ-Jβ	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLE（SEQ ID NO.10）
			11	ST18G02Cβ	DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG（SEQ ID NO.11）
12	ST18G02 beta chain	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG（SEQ ID NO.12）
			13	ST18G02P2A	GSGATNFSLLKQAGDVEENPGP（SEQ ID NO.13）
14	ST18G02 full Length	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGPMDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.14）
			15	Murine ST18G02 alpha-CDR 1	NTAFDY（SEQ ID NO.15）
16	Murine ST18G02 alpha-CDR 2	IRPDVSE（SEQ ID NO.16）
			17	Murine ST18G02 alpha-CDR 3	CAAPGEDSNYQLIW（SEQ ID NO.17）
18	Murine ST18G02 beta-CDR 1	MNHEY（SEQ ID NO.18）
			19	Murine ST18G02 beta-CDR 2	SMNVEV（SEQ ID NO.19）
20	Murine ST18G02 beta-CDR 3	CASSLSVGDTQYF（SEQ ID NO.20）
			21	Murine ST18G02V alpha-J alpha	MDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPD（SEQ ID NO.21）
22	Murine ST18G02C alpha	IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.22）
			23	Murine ST18G02 alpha chain	MDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.23）
24	Murine ST18G02V beta-J beta	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLE（SEQ ID NO.24）
			25	Murine ST18G02C beta	DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS（SEQ ID NO.25）
26	Murine ST18G02 beta chain	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS（SEQ ID NO.26）
			27	Murine ST18G02P2A	GSGATNFSLLKQAGDVEENPGP（SEQ ID NO.27）
28	Murine ST18G02 full Length	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSGSGATNFSLLKQAGDVEENPGPMDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.28）
			29	Murine + sequence optimization ST18G02 alpha CDR1	NTAFDY（SEQ ID NO.29）
30	Murine + sequence optimization ST18G02 alpha CDR2	IRPDVSE（SEQ ID NO.30）
			31	Murine + sequence optimization ST18G02 alpha CDR3	CAAPGEDSNYQLIW（SEQ ID NO.31）
32	Murine + sequence optimization ST18G02 beta-CDR 1	MNHEY（SEQ ID NO.32）
			33	Murine + sequence optimization ST18G02 beta-CDR 2	SMNVEV（SEQ ID NO.33）
34	Murine + sequence optimization ST18G02 beta-CDR 3	CASSLSVGDTQYF（SEQ ID NO.34）
			35	Murine + sequence optimization ST18G02vα -jα	MDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPD（SEQ ID NO.35）
36	Murine + sequence optimization ST18G02cα	IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.36）
			37	Murine + sequence optimization ST18G02 alpha chain	MDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.37）
38	Murine + sequence optimization ST18G02vβ -jβ	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLE（SEQ ID NO.38）
			39	Murine + sequence optimization ST18G02cβ	DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS（SEQ ID NO.39）
40	Murine + sequence optimization ST18G02 beta chain	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS（SEQ ID NO.40）
			41	Murine+sequence optimization ST18G02P2A	GSGATNFSLLKQAGDVEENPGP（SEQ ID NO.41）
42	Murine + sequence optimization ST18G02 full length	MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLSVGDTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSGSGATNFSLLKQAGDVEENPGPMDKILGASFLVLWLQLCWVSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSLHIMDSQPGDSATYFCAAPGEDSNYQLIWGAGTKLIIKPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS（SEQ ID NO.42）
			43	ST18G02α-CDR1 DNA	AACACTGCGTTTGACTAC（SEQ ID NO.43）
44	ST18G02α-CDR2 DNA	ATACGTCCAGATGTGAGTGAA（SEQ ID NO.44）
			45	ST18G02α-CDR3 DNA	TGTGCAGCTCCCGGGGAGGATAGCAACTATCAGTTAATCTGG（SEQ ID NO.45）
46	ST18G02β-CDR1 DNA	ATGAACCATGAGTAT（SEQ ID NO.46）
			47	ST18G02β-CDR2 DNA	TCAATGAATGTTGAGGTG（SEQ ID NO.47）
48	ST18G02β-CDR3 DNA	TGTGCCAGCAGTTTATCAGTAGGAGATACGCAGTATTTT（SEQ ID NO.48）
			49	ST18G02Vα-Jα DNA	ATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTATGCTGGGTGAGTGGCCAACAGAAGGAGAAAAGTGACCAGCAGCAGGTGAAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATTATAAACTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTACCAACAATTCCCTGGGAAAGGCCCTGCATTATTGATAGCCATACGTCCAGATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAAAGTGCCAAGCAGTTCTCATTGCATATCATGGATTCCCAGCCTGGAGACTCAGCCACCTACTTCTGTGCAGCTCCCGGGGAGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGAT（SEQ ID NO.49）
50	ST18G02Vβ-Jβ DNA	ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTATCAGTAGGAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG（SEQ ID NO.50）
			51	ST18G02Cα DNA	ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA（SEQ ID NO.51）
52	ST18G02Cβ DNA	GACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC（SEQ ID NO.52）
			53	ST18G02P2A DNA	GGAAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCC（SEQ ID NO.53）
54	ST18G02 full-length DNA	ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTATCAGTAGGAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCGGAAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTATGCTGGGTGAGTGGCCAACAGAAGGAGAAAAGTGACCAGCAGCAGGTGAAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATTATAAACTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTACCAACAATTCCCTGGGAAAGGCCCTGCATTATTGATAGCCATACGTCCAGATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAAAGTGCCAAGCAGTTCTCATTGCATATCATGGATTCCCAGCCTGGAGACTCAGCCACCTACTTCTGTGCAGCTCCCGGGGAGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA（SEQ ID NO.54）
			55	Murine ST18G02 alpha-CDR 1 DNA	AACACTGCGTTTGACTAC（SEQ ID NO.55）
56	Murine ST18G02 alpha-CDR 2 DNA	ATACGTCCAGATGTGAGTGAA（SEQ ID NO.56）
			57	Murine ST18G02 alpha-CDR 3 DNA	TGTGCAGCTCCCGGGGAGGATAGCAACTATCAGTTAATCTGG（SEQ ID NO.57）
58	Murine ST18G02 beta-CDR 1 DNA	ATGAACCATGAGTAT（SEQ ID NO.58）
			59	Murine ST18G02 beta-CDR 2 DNA	TCAATGAATGTTGAGGTG（SEQ ID NO.59）
60	Murine ST18G02 beta-CDR 3 DNA	TGTGCCAGCAGTTTATCAGTAGGAGATACGCAGTATTTT（SEQ ID NO.60）
			61	Murine ST18G02V alpha-J alpha DNA	ATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTATGCTGGGTGAGTGGCCAACAGAAGGAGAAAAGTGACCAGCAGCAGGTGAAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATTATAAACTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTACCAACAATTCCCTGGGAAAGGCCCTGCATTATTGATAGCCATACGTCCAGATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAAAGTGCCAAGCAGTTCTCATTGCATATCATGGATTCCCAGCCTGGAGACTCAGCCACCTACTTCTGTGCAGCTCCCGGGGAGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGAT（SEQ ID NO.61）
62	Murine ST18G02V beta-J beta DNA	ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTATCAGTAGGAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG（SEQ ID NO.62）
			63	Murine ST18G02C alpha DNA	ATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGCTGGTTATGGTTCTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA（SEQ ID NO.63）
64	Murine ST18G02C beta DNA	GATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAAAATTCA（SEQ ID NO.64）
			65	Murine ST18G02P2A DNA	GGAAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCC（SEQ ID NO.65）
66	Murine ST18G02 full-length DNA	ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTATCAGTAGGAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAAAATTCAGGAAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTATGCTGGGTGAGTGGCCAACAGAAGGAGAAAAGTGACCAGCAGCAGGTGAAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATTATAAACTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTACCAACAATTCCCTGGGAAAGGCCCTGCATTATTGATAGCCATACGTCCAGATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAAAGTGCCAAGCAGTTCTCATTGCATATCATGGATTCCCAGCCTGGAGACTCAGCCACCTACTTCTGTGCAGCTCCCGGGGAGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGATATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGCTGGTTATGGTTCTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA（SEQ ID NO.66）
			67	Murine + sequence optimization ST18G02 alpha-CDR 1 DNA	AACACCGCCTTCGACTAC（SEQ ID NO.67）
68	Murine + sequence optimization ST18G02 alpha-CDR 2 DNA	ATCAGACCCGACGTGAGCGAG（SEQ ID NO.68）
			69	Murine + sequence optimization ST18G02 alpha-CDR 3 DNA	TGCGCCGCCCCTGGCGAGGACAGCAACTATCAGCTGATCTGG（SEQ ID NO.69）
70	Murine + sequence optimization ST18G02 beta-CDR 1 DNA	ATGAACCACGAGTAC（SEQ ID NO.70）
			71	Murine + sequence optimization ST18G02 beta-CDR 2 DNA	AGCATGAACGTGGAGGTG（SEQ ID NO.71）
72	Murine + sequence optimization ST18G02 beta-CDR 3 DNA	TGCGCTAGCAGCCTGAGCGTGGGCGACACACAGTACTTC（SEQ ID NO.72）
			73	Murine + sequence optimization ST18G02V alpha-J alpha DNA	ATGGACAAGATCCTGGGCGCTAGCTTCCTGGTGCTGTGGCTGCAGCTGTGCTGGGTGAGCGGGCAGCAGAAGGAGAAGAGCGATCAGCAACAAGTGAAGCAGAGCCCTCAGAGCCTGATCGTGCAGAAGGGCGGCATCAGCATCATCAACTGCGCCTACGAGAACACCGCCTTCGACTACTTCCCCTGGTATCAGCAGTTCCCCGGCAAGGGCCCCGCCCTGCTGATCGCCATCAGACCCGACGTGAGCGAGAAGAAGGAGGGCAGATTCACCATCAGCTTCAACAAGAGCGCCAAGCAGTTCAGCCTGCACATCATGGACAGCCAACCCGGCGACAGCGCCACCTACTTCTGCGCCGCCCCTGGCGAGGACAGCAACTATCAGCTGATCTGGGGCGCCGGCACCAAGCTGATCATCAAGCCCGAC（SEQ ID NO.73）
74	Murine + sequence optimization ST18G02 ca DNA	ATTCAGAACCCCGAGCCCGCCGTGTATCAGCTGAAGGACCCTAGAAGCCAAGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAAATCAACGTGCCCAAGACCATGGAGAGCGGCACCTTCATCACCGACAAGACCGTGCTGGACATGAAGGCCATGGACAGCAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACAAGCTTCACCTGCCAAGACATCTTCAAGGAGACCAACGCCACCTACCCTAGCAGCGACGTGCCCTGCGACGCCACCCTGACCGAGAAGAGCTTCGAGACCGACATGAACCTGAACTTTCAGAACCTGCTGGTGATGGTGCTGAGAATCCTGCTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGAGCAGCTGA（SEQ ID NO.74）
			75	Murine + sequence optimization ST18G02V beta-J beta DNA	ATGGGCCCTCAGCTCCTGGGGTACGTGGTGCTGTGCCTCCTGGGGGCCGGCCCTCTCGAGGCCCAAGTGACACAGAACCCCCGGTACCTGATCACCGTGACCGGCAAGAAGCTGACCGTGACCTGCAGCCAAAACATGAACCACGAGTACATGAGCTGGTACAGACAAGACCCCGGCCTGGGCCTGAGACAGATCTACTACAGCATGAACGTGGAGGTGACCGACAAGGGCGACGTGCCCGAGGGCTACAAGGTGAGCAGAAAGGAGAAGAGAAACTTCCCCCTGATCCTGGAGAGCCCTAGCCCCAACCAAACAAGCCTGTACTTCTGCGCTAGCAGCCTGAGCGTGGGCGACACACAGTACTTCGGCCCCGGCACAAGACTGACAGTGCTGGAA（SEQ ID NO.75）
76	Murine + sequence optimization ST18G02cβ DNA	GATCTGAGAAACGTGACCCCCCCCAAGGTGAGCCTGTTCGAGCCTAGCAAGGCCGAGATCGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCTAGAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTGAGCACCGACCCCCAAGCCTACAAGGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGAGAGTGAGCGCCACCTTCTGGCACAACCCTAGAAACCACTTCAGATGCCAAGTGCAGTTCCACGGCCTGAGCGAGGAGGACAAGTGGCCCGAGGGCAGCCCCAAGCCCGTCACACAGAACATCAGCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCATCACAAGCGCTAGCTATCAGCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTCTATGCCGTGCTGGTGAGCACCCTGGTGGTGATGGCCATGGTGAAGAGAAAGAACAGC（SEQ ID NO.76）
			77	Murine+sequence optimization ST18G02P2A DNA	GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAACCCCGGCCCC（SEQ ID NO.77）
78	Murine + sequence optimization ST18G02 full-length DNA	ATGGGCCCTCAGCTCCTGGGGTACGTGGTGCTGTGCCTCCTGGGGGCCGGCCCTCTCGAGGCCCAAGTGACACAGAACCCCCGGTACCTGATCACCGTGACCGGCAAGAAGCTGACCGTGACCTGCAGCCAAAACATGAACCACGAGTACATGAGCTGGTACAGACAAGACCCCGGCCTGGGCCTGAGACAGATCTACTACAGCATGAACGTGGAGGTGACCGACAAGGGCGACGTGCCCGAGGGCTACAAGGTGAGCAGAAAGGAGAAGAGAAACTTCCCCCTGATCCTGGAGAGCCCTAGCCCCAACCAAACAAGCCTGTACTTCTGCGCTAGCAGCCTGAGCGTGGGCGACACACAGTACTTCGGCCCCGGCACAAGACTGACAGTGCTGGAAGATCTGAGAAACGTGACCCCCCCCAAGGTGAGCCTGTTCGAGCCTAGCAAGGCCGAGATCGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCTAGAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTGAGCACCGACCCCCAAGCCTACAAGGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGAGAGTGAGCGCCACCTTCTGGCACAACCCTAGAAACCACTTCAGATGCCAAGTGCAGTTCCACGGCCTGAGCGAGGAGGACAAGTGGCCCGAGGGCAGCCCCAAGCCCGTCACACAGAACATCAGCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCATCACAAGCGCTAGCTATCAGCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTCTATGCCGTGCTGGTGAGCACCCTGGTGGTGATGGCCATGGTGAAGAGAAAGAACAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGACAAGATCCTGGGCGCTAGCTTCCTGGTGCTGTGGCTGCAGCTGTGCTGGGTGAGCGGGCAGCAGAAGGAGAAGAGCGATCAGCAACAAGTGAAGCAGAGCCCTCAGAGCCTGATCGTGCAGAAGGGCGGCATCAGCATCATCAACTGCGCCTACGAGAACACCGCCTTCGACTACTTCCCCTGGTATCAGCAGTTCCCCGGCAAGGGCCCCGCCCTGCTGATCGCCATCAGACCCGACGTGAGCGAGAAGAAGGAGGGCAGATTCACCATCAGCTTCAACAAGAGCGCCAAGCAGTTCAGCCTGCACATCATGGACAGCCAACCCGGCGACAGCGCCACCTACTTCTGCGCCGCCCCTGGCGAGGACAGCAACTATCAGCTGATCTGGGGCGCCGGCACCAAGCTGATCATCAAGCCCGACATTCAGAACCCCGAGCCCGCCGTGTATCAGCTGAAGGACCCTAGAAGCCAAGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAAATCAACGTGCCCAAGACCATGGAGAGCGGCACCTTCATCACCGACAAGACCGTGCTGGACATGAAGGCCATGGACAGCAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACAAGCTTCACCTGCCAAGACATCTTCAAGGAGACCAACGCCACCTACCCTAGCAGCGACGTGCCCTGCGACGCCACCCTGACCGAGAAGAGCTTCGAGACCGACATGAACCTGAACTTTCAGAACCTGCTGGTGATGGTGCTGAGAATCCTGCTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGAGCAGCTGA（SEQ ID NO.78）
			79	ST18G02 alpha-CDR 3 optimized amino acid sequence (ST 18G02-AM 12)	CASSLSVGATQYF（SEQ ID NO.79）
80	ST18G02 alpha-CDR 3 optimized amino acid sequence (ST 18G02-AM 17)	CASSLSVGDTQSF（SEQ ID NO.80）
			81	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 26)	CASSLSVGATQSF（SEQ ID NO.81）
82	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 31)	CASSLSVADTQSF（SEQ ID NO.82）
			83	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 40)	CASSLSVADTQYF（SEQ ID NO.83）
84	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 41)	CASSLSVASVQYF（SEQ ID NO.84）
			85	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 43)	CASSLSVATVQYF（SEQ ID NO.85）
86	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 54)	CASSLSVAITQYF（SEQ ID NO.86）
			87	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 57)	CASSLSVAMTQYF（SEQ ID NO.87）
88	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 60)	CASSLSVASTQYF（SEQ ID NO.88）
			89	ST18G02 beta-CDR 3 optimized amino acid sequence (ST 18G02-AM 81)	CASSLSVAAVQYF（SEQ ID NO.89）
90	ST18G02 alpha-CDR 3 optimized nucleotide sequence (ST 18G02-AM 12)	TGCGCTAGCAGCCTGAGCGTGGGCGCCACACAGTACTTC（SEQ ID NO.90）
			91	ST18G02 alpha-CDR 3 optimized nucleotide sequence (ST 18G02-AM 17)	TGCGCTAGCAGCCTGAGCGTGGGCGACACACAGAGCTTC（SEQ ID NO.91）
92	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 26)	TGCGCTAGCAGCCTGAGCGTGGGCGCCACACAGAGCTTC（SEQ ID NO.92）
			93	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 31)	TGCGCTAGCAGCCTGAGCGTGGCCGACACACAGAGCTTC（SEQ ID NO.93）
94	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 40)	TGCGCTAGCAGCCTGAGCGTGGCCGACACACAGTACTTC（SEQ ID NO.94）
			95	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 41)	TGCGCTAGCAGCCTGAGCGTGGCCAGCGTGCAGTACTTC（SEQ ID NO.95）
96	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 43)	TGCGCTAGCAGCCTGAGCGTGGCCACCGTGCAGTACTTC（SEQ ID NO.96）
			97	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 54)	TGCGCTAGCAGCCTGAGCGTGGCCATCACACAGTACTTC（SEQ ID NO.97）
98	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 57)	TGCGCTAGCAGCCTGAGCGTGGCCATGACACAGTACTTC（SEQ ID NO.98）
			99	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 60)	TGCGCTAGCAGCCTGAGCGTGGCCAGCACACAGTACTTC（SEQ ID NO.99）
100	ST18G02 beta-CDR 3 optimized nucleotide sequence (ST 18G02-AM 81)	TGCGCTAGCAGCCTGAGCGTGGCCGCCGTGCAGTACTTC（SEQ ID NO.100）
			101	MAGE-A4146-154	RCFPVIFGK（SEQ ID NO.101）

Claims

1. An isolated tumor-specific T Cell Receptor (TCR), or antigen binding domain thereof, that specifically binds to a major tissue compatibility complex (MHC)/MAGE-A4 epitope complex, the tumor-specific TCR comprising: a TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 6.

2. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1, wherein said MAGE-A4 epitope is the amino acid sequence set forth in SEQ ID No. 101.

3. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1 or 2, wherein said MHC is HLA-A x 11:01.

4. An isolated tumor-specific T Cell Receptor (TCR) or antigen-binding domain thereof that specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1, comprising: a TCR alpha chain comprising the variable region of the amino acid sequence of SEQ ID NO. 7, and a TCR beta chain comprising the variable region of the amino acid sequence of SEQ ID NO. 10.

5. An isolated tumor-specific T Cell Receptor (TCR) or antigen-binding domain thereof that specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1, comprising: a TCR alpha chain comprising the amino acid sequences of SEQ ID NOs 7 and 8, and a TCR beta chain comprising the amino acid sequences of SEQ ID NOs 10 and 11.

6. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1, which is isolated or purified or recombinant.

7. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1, which is chimeric, humanized or human.

8. An isolated tumor-specific T Cell Receptor (TCR) or antigen-binding domain thereof that specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 1, wherein the TCR or antigen-binding domain thereof is single chain.

9. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 8, wherein the TCR a chain further comprises an a constant (ca) region and/or the TCR β chain further comprises a β constant (cβ) region.

10. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 9, wherein the cα region and the cβ region are mouse constant regions.

11. An isolated tumor specific T Cell Receptor (TCR) or antigen binding domain thereof which specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 10, wherein the ca region comprises the amino acid sequence of SEQ ID No (ca) 22 and/or the ca region comprises the amino acid sequence of SEQ ID No (ca) 25.

12. An isolated tumor-specific T Cell Receptor (TCR) or antigen-binding domain thereof that specifically binds to the major tissue compatibility complex (MHC)/MAGE-A4 epitope complex according to claim 11, comprising: a TCR alpha chain comprising the amino acid sequences of SEQ ID NOs: 21 and 22, and a TCR beta chain comprising the amino acid sequences of SEQ ID NOs:24 and 25.

13. The isolated TCR or antigen-binding domain thereof according to claim 1, wherein the TCR alpha chain and/or the TCR beta chain further comprises a signal peptide.

14. An isolated nucleic acid molecule encoding a TCR a chain and a TCR β chain of a tumor-specific T cell receptor or antigen-binding domain thereof according to any one of claims 1-13.

15. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid sequence of the nucleic acid molecule is codon optimized.

16. The isolated nucleic acid molecule of claim 14 or 15, wherein the nucleotide sequence encoding the TCR a chain and the nucleotide sequence encoding the TCR β chain are separated by a self-cleaving peptide sequence.

17. The isolated nucleic acid molecule of claim 16, wherein the self-cleaving peptide is P2A comprising the nucleotide sequence set forth in SEQ ID nos. 53.

18. An engineered immune cell comprising the isolated TCR or antigen-binding domain thereof, or nucleic acid encoding the same, of any one of claims 1-13, which is a cell line or a primary or heterologous cell obtained from a subject.

19. The engineered immune cell of claim 18, wherein the immune cell is a human cell comprising a T cell, NK cell, macrophage.

20. The engineered immune cell of claim 19, wherein the immune cell is a T cell.

21. The engineered immune cell of claim 20, wherein the engineered immune cell comprises a CD8 ⁺ T cell.

22. The engineered immune cell of claim 20, wherein the engineered immune cell comprises a CD4 ⁺ T cell.

23. A pharmaceutical composition comprising the engineered immune cell of claim 21 or 22, and a pharmaceutically acceptable carrier.

24. Use of the engineered immune cell of claim 18 in the manufacture of a medicament for the treatment of MAGE-A4 expressing cancer;

The cancers include synovial sarcoma, myxotype liposarcoma, non-small cell lung cancer, head and neck squamous carcinoma, ovarian cancer, urothelial cell carcinoma, melanoma, and gastroesophageal cancer.

25. The use of claim 24, wherein the engineered immune cell is an autologous or allogeneic cell of the subject.

26. An isolated tumor-specific T Cell Receptor (TCR) or antigen binding domain thereof that specifically binds to a major tissue compatibility complex (MHC)/MAGE-A4 epitope complex, wherein the tumor-specific TCR comprises: a TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 79.

27. An isolated tumor-specific T Cell Receptor (TCR) or antigen binding domain thereof that specifically binds to a major tissue compatibility complex (MHC)/MAGE-A4 epitope complex, wherein the tumor-specific TCR comprises: a TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 80.

28. An isolated tumor-specific T Cell Receptor (TCR) or antigen binding domain thereof that specifically binds to a major tissue compatibility complex (MHC)/MAGE-A4 epitope complex, wherein the tumor-specific TCR comprises: a TCR a chain comprising CDR1 of the amino acid sequence of SEQ ID No. 1, CDR2 of the amino acid sequence of SEQ ID No. 2, and CDR3 of the amino acid sequence of SEQ ID No. 3; and a TCR beta chain comprising CDR1 of the amino acid sequence of SEQ ID NO. 4, CDR2 of the amino acid sequence of SEQ ID NO. 5 and CDR3 of the amino acid sequence of SEQ ID NO. 83.