MAGE-a1-specific t cell receptors and uses thereof

Abstract

The present invention provides a tumor-specific T cell receptor that specifically binds a major histocompatibility complex (MHC) / MAGE-A1 epitope complex, engineered T cells comprising the receptor, and methods of using the same to treat diseases, including cancer.

Description

[0001] This case is a divisional application of the application filed on April 27, 2022, entitled "MAGE-A1 Specific T-cell Receptor and Its Use Thereof", with application number CN202210449926.3.

[0002] Submitting sequence lists as ASCII text files

[0003] The following content, submitted as an ASCII text file, is incorporated herein by reference in its entirety: Computer-readable form of sequence listing (CRF) (filename: 776902000743SEQLIST.xml, record date: September 1, 2022, size: 540334B). Technical Field

[0004] This application relates to the field of cancer immunotherapy. Specifically, in some aspects, this application relates to binding molecules, such as complexes capable of specifically binding major histocompatibility complex (MHC) molecules and MAGE-A1 peptides. In particular, this application relates to T-cell receptors (TCRs) that bind to or recognize such peptide epitopes, including their antigen-binding domains. This application further relates to engineered cells comprising such binding molecules, such as TCRs, and their use in adoptive cell therapy. Background Technology

[0005] The human body possesses a complex immune system that protects it from diseases, including malignant tumors. The innate immune response to tumors is typically triggered by tumor antigens, which include mutated proteins specifically expressed in cancer cells and tumor-associated antigens (TAAs) overexpressed in cancer-originating tissues but not fully recognized as "self." Antigen-presenting cells (APCs) (especially dendritic cells (DCs)) encountering tumor antigens process them and present them on their cell surface. Upon maturation, tumor antigen-loaded DCs elicit a T-cell response to cancer cells harboring the tumor antigens, involving cytotoxic T cells, helper T cells, and functionally distinct effector T cells and memory T cells. Cytotoxic T cells can specifically bind to and recognize cancer cells expressing tumor antigens via their surface T-cell receptors (TCRs) and kill cancer cells by releasing cytokines, enzymes, and cytotoxins, or by triggering a pro-apoptotic signaling cascade through cell-cell interactions.

[0006] Adoptive lymphocyte therapy (ACT) offers a promising concept for cancer treatment by administering ex vivo expanded lymphocytes to a subject. For example, tumor-infiltrating lymphocytes (TILs) can be isolated from a patient's tumor, expanded ex vivo, and then infused back into the patient after removing the patient's native non-myeloid lymphocytes. However, the variability in the affinity of TILs isolated from each patient or donor limits antitumor efficacy in clinical trials. Furthermore, most antigen-specific mutations that trigger an immune response are found only in an individual's cancer, rather than in multiple patients.

[0007] Engineered T cells containing tumor antigen-specific T-cell receptors (TCRs) can overcome some of the challenges currently faced by follow-up lymphocyte therapy because they can rapidly generate tumor-reactive T lymphocytes with defined antigen specificity. There is a need in this field for TCRs that target different tumor antigens and provide high in vivo anti-tumor efficacy and low off-target effects. Common problems with TCR-T cell therapy include serious adverse events in clinical trials (such as central nervous system toxicity), which may be related to inappropriate target selection (so-called on-target / off-tumor effects) and biased expansion of the T-cell population.

[0008] Melanoma-associated antigen A1 (MAGE-A1) is a cancer-testis antigen found in adult male germ cells and embryonic cells, playing a crucial role in tumor detection and immunotherapy. The MAGE-A1 gene belongs to the MAGE family, which can be divided into two classes: class I and class II. Class I, located on the X chromosome, is an important component of tumor-associated antigens and includes MAGE-A, MAGE-B, and MAGE-C. The MAGE-A1 gene is expressed only in genomically unstable tissues prone to demethylation, such as normal testicular tissue and various tumor tissues, and is considered closely related to tumor progression and poor prognosis. Research indicates that tumors and / or tumor-derived factors in genetically susceptible individuals may trigger myopathy, and immune responses to myositis antigens may be related to initial or subclinical antitumor responses in patients. Therefore, MAGE-A1-specific TCRs and TCR-Ts hold promise as cancer therapies with high antitumor efficacy and low off-target effects. Invention Overview

[0009] According to a first aspect of the invention, a tumor-specific T-cell receptor (TCR) or its antigen-binding domain that specifically binds to the major tissue compatibility complex (MHC) / MAGE-A1 epitope complex is provided, said tumor-specific TCR comprising:

[0010] (1) A TCRα 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 TCRβ 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;

[0011] (2) A TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:43, CDR2 having the amino acid sequence of SEQ ID NO:44, and CDR3 having the amino acid sequence of SEQ ID NO:45; and a TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO:46, CDR2 having the amino acid sequence of SEQ ID NO:47, and CDR3 having the amino acid sequence of SEQ ID NO:48;

[0012] (3) A TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:85, CDR2 having the amino acid sequence of SEQ ID NO:86, and CDR3 having the amino acid sequence of SEQ ID NO:87; and a TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO:88, CDR2 having the amino acid sequence of SEQ ID NO:89, and CDR3 having the amino acid sequence of SEQ ID NO:90;

[0013] (4) A TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:127, CDR2 having the amino acid sequence of SEQ ID NO:128, and CDR3 having the amino acid sequence of SEQ ID NO:129; and a TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO:130, CDR2 having the amino acid sequence of SEQ ID NO:131, and CDR3 having the amino acid sequence of SEQ ID NO:132; or

[0014] (5) A TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO:169, CDR2 having the amino acid sequence of SEQ ID NO:170, and CDR3 having the amino acid sequence of SEQ ID NO:171; and a TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO:172, CDR2 having the amino acid sequence of SEQ ID NO:173, and CDR3 having the amino acid sequence of SEQ ID NO:174.

[0015] In some embodiments, an isolated tumor-specific TCR or its antigen-binding domain that specifically binds to the major tissue compatibility complex (MHC) / MAGE-A1 epitope complex is provided, wherein the MAGE-A1 epitope comprises the amino acid sequence of SEQ ID NOs: 365, 366, 367, 368 or 369.

[0016] In some embodiments according to any of the above, the MHC is HLA-DRA*01:01 / DRB1*12:02 or DRA*01:01 / DRB1*07:01.

[0017] In some embodiments according to any of the foregoing, the isolated tumor-specific TCR or its antigen-binding domain comprises:

[0018] (1) A TCRα chain containing a variable region having the amino acid sequence of SEQ ID NO:7 and a TCRβ chain containing a variable region having the amino acid sequence of SEQ ID NO:10;

[0019] (2) A TCRα chain containing a variable region having the amino acid sequence of SEQ ID NO:49 and a TCRβ chain containing a variable region having the amino acid sequence of SEQ ID NO:52;

[0020] (3) A TCRα chain containing a variable region having the amino acid sequence of SEQ ID NO:91 and a TCRβ chain containing a variable region having the amino acid sequence of SEQ ID NO:94;

[0021] (4) A TCRα chain containing a variable region having the amino acid sequence of SEQ ID NO: 133 and a TCRβ chain containing a variable region having the amino acid sequence of SEQ ID NO: 136; or

[0022] (5) A TCRα chain containing a variable region having the amino acid sequence of SEQ ID NO:175 and a TCRβ chain containing a variable region having the amino acid sequence of SEQ ID NO:178.

[0023] In some embodiments according to any of the foregoing, the isolated tumor-specific TCR or its antigen-binding domain includes:

[0024] (1) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:7 and 8 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:10 and 11 or an amino acid sequence having at least 90% sequence identity with it.

[0025] (2) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:49 and 50 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:52 and 53 or an amino acid sequence having at least 90% sequence identity with it.

[0026] (3) A TCRα chain containing amino acid sequences having SEQ ID NOs:91 and 92 or amino acid sequences having at least 90% sequence identity with them, and a TCRβ chain containing amino acid sequences having SEQ ID NOs:94 and 95 or amino acid sequences having at least 90% sequence identity with them.

[0027] (4) A TCRα chain comprising an amino acid sequence having SEQ ID NOs: 133 and 134 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs: 135 and 136 or an amino acid sequence having at least 90% sequence identity with it; or

[0028] (5) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:175 and 176 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:178 and 179 or an amino acid sequence having at least 90% sequence identity with it.

[0029] The isolated TCR or its antigen-binding domain, as described in any of the embodiments of the above, is isolated, purified, or recombinant.

[0030] The isolated TCR or its antigen-binding domain, as described in some of the embodiments of the above, is chimeric, humanized, or human.

[0031] The isolated TCR or its antigen-binding domain as described in some of the embodiments of the above, wherein the TCR or its antigen-binding domain is single-stranded.

[0032] According to embodiments of any of the above, the isolated TCR or its antigen-binding domain further comprises an α constant (Cα) region and / or the TCR β chain further comprises a β constant (Cβ) region.

[0033] The isolated TCR or its antigen-binding domain as described in some of the embodiments of the above, wherein the Cα region and the Cβ region are mouse constant regions.

[0034] According to embodiments of any of the above, the isolated TCR or its antigen-binding domain comprises an amino acid sequence of SEQ ID NOs:22, 64, 106, 148 or 190 or an amino acid sequence having at least 90% sequence identity with it, and / or the Cβ region comprises an amino acid sequence of SEQ ID NOs:25, 67, 109, 151 or 193 or an amino acid sequence having at least 90% sequence identity with it.

[0035] The isolated TCR or its antigen-binding domain according to embodiments of any of the above includes:

[0036] (1) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:21 and 22 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:24 and 25 or an amino acid sequence having at least 90% sequence identity with it.

[0037] (2) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:63 and 64 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:66 and 67 or an amino acid sequence having at least 90% sequence identity with it.

[0038] (3) A TCRα chain containing amino acid sequences having SEQ ID Nos: 105 and 106 or amino acid sequences having at least 90% sequence identity with them, and a TCRβ chain containing amino acid sequences having SEQ ID Nos: 108 and 109 or amino acid sequences having at least 90% sequence identity with them.

[0039] (4) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:147 and 148 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:149 and 150 or an amino acid sequence having at least 90% sequence identity with it; or

[0040] (5) A TCRα chain comprising an amino acid sequence having SEQ ID NOs:189 and 190 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain comprising an amino acid sequence having SEQ ID NOs:192 and 193 or an amino acid sequence having at least 90% sequence identity with it.

[0041] According to embodiments of any of the above, the isolated TCR or its antigen-binding domain further comprises a signal peptide.

[0042] In some embodiments, an isolated nucleic acid is provided that encodes an isolated TCR or its antigen-binding domain or its TCRα or TCRβ chain as described in some of the embodiments described above.

[0043] The isolated nucleic acid molecule according to the implementation scheme of any of the above, wherein the nucleic acid sequence is codon optimized.

[0044] The isolated nucleic acid molecule according to the embodiments of any of the above, wherein the nucleotide sequence encoding the TCRα chain and the nucleotide sequence encoding the TCRβ chain are separated by a self-splitting peptide sequence.

[0045] The isolated nucleic acid molecule according to the embodiments of any of the above, wherein the self-splitting peptide is P2A, comprising the amino acid sequence shown in SEQ ID NO:13.

[0046] In some embodiments, isolated nucleic acids are provided that encode the TCRα and TCRβ chains of the tumor-specific TCR.

[0047] In some embodiments, engineered immune cells are provided, the engineered immune cells comprising a tumor-specific TCR or its encoding nucleic acid as described in any of the embodiments above, the engineered cells being a cell line or primary cells obtained from a subject, preferably human cells.

[0048] The engineered immune cells described in the implementation scheme of any of the above, wherein the immune cells are T cells.

[0049] The engineered cells according to the embodiments of any of the above, wherein the engineered cells comprise CD8+ T cells.

[0050] The engineered cells according to the embodiments of any of the above, wherein the engineered cells comprise CD4+ T cells.

[0051] In some embodiments, a pharmaceutical composition is provided comprising engineered immune cells as described in any of the embodiments above, and a pharmaceutically acceptable carrier.

[0052] In some embodiments, a method for treating a disease or condition expressing MAGE-A1 is provided, comprising administering to a subject in need an effective amount of the pharmaceutical composition according to any of the embodiments described above.

[0053] The method described in any of the above embodiments is cancer.

[0054] The method according to any of the above embodiments, wherein the cancer is selected from lung cancer, liver cancer, skin cancer, breast cancer, and head and neck tumors.

[0055] According to the method described in any of the above embodiments, the engineered cells are the subject's own cells.

[0056] The method described in any of the above embodiments further includes administering one or more of the following to the subject: immunosuppressants, therapeutic antibodies, chemotherapy, radiation therapy, surgery, or any combination thereof. Attached Figure Description

[0057] The following embodiments are intended purely as examples of this patent application and should not be construed as limiting the invention in any way. The following embodiments and detailed descriptions are provided by way of illustration and not by way of limitation.

[0058] Figure 1A Showing five example MAGE-A1-specific TCRs, their antigen recognition, and the MHCs that present the antigen.

[0059] Figure 1B Displaying MAGE-A1-specific TCR constructions (wild-type, murine, and murine sequence-optimized constructs).

[0060] Figure 2A-2C Screening and characterization of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01C05.

[0061] Figure 3 This demonstrates epitope recognition on T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01C05.

[0062] Figure 4 HLA restriction assay of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01C05 in the K562 cell line.

[0063] Figure 5This demonstrates the cytotoxicity of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01C05 against the 293T cell line that presents the MAGE-A1 peptide via DRA*01:01 / DRB1*12:02 in vitro.

[0064] Figure 6 This demonstrates the cytotoxicity of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01C05 against the 293T cell line that presents MAGE-A1 protein via DRA*01:01 / DRB1*12:02 in vitro.

[0065] Figure 7 This demonstrates the cytotoxicity of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01C05 against the A549 cell line that presents MAGE-A1 protein via DRA*01:01 / DRB1*12:02 in vitro.

[0066] Figures 8A-8C Screening and characterization of T cells expressing the MAGE-A1 antigen-specific T cell receptor 35C11.

[0067] Figure 9 This demonstrates epitope recognition on T cells expressing the MAGE-A1 antigen-specific T cell receptor 35C11.

[0068] Figure 10 HLA restriction assays of T cells expressing the MAGE-A1 antigen-specific T cell receptor 35C11 in the K562 cell line.

[0069] Figure 11 This study demonstrates the cytotoxicity of T cells expressing the MAGE-A1 antigen-specific T cell receptor 35C11 against the 293T cell line that presents the MAGE-A1 peptide via DRA*01:01 / DRB1*07:01 in vitro.

[0070] Figures 12A-12C Screening and characterization of T cells expressing the MAGE-A1 antigen-specific T cell receptor 35D09.

[0071] Figure 13 This demonstrates epitope recognition on T cells expressing the MAGE-A1 antigen-specific T cell receptor 35D09.

[0072] Figure 14 HLA restriction assays of T cells expressing the MAGE-A1 antigen-specific T cell receptor 35D09 in the K562 cell line.

[0073] Figure 15This study demonstrates the cytotoxicity of T cells expressing the MAGE-A1 antigen-specific T cell receptor 35D09 against the 293T cell line that presents the MAGE-A1 peptide via DRA*01:01 / DRB1*07:01 in vitro.

[0074] Figures 16A-16C Screening and characterization of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01G05.

[0075] Figure 17 This demonstrates epitope recognition on T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01G05.

[0076] Figure 18 HLA restriction assays of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01G05 in the K562 cell line.

[0077] Figure 19 This demonstrates the cytotoxicity of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01G05 against the 293T cell line that presents the MAGE-A1 peptide via DRA*01:01 / DRB1*07:01 in vitro.

[0078] Figures 20A-20C Screening and characterization of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01F06.

[0079] Figure 21 This demonstrates epitope recognition on T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01F06.

[0080] Figure 22 The HLA blocking antibody test showed that T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01F06 were restricted to HLA-DR.

[0081] Figure 23 HLA restriction assay of T cells expressing the MAGE-A1 antigen-specific T cell receptor ST01F06 in the K562 cell line. Invention Details

[0082] I. Definition

[0083] Before describing the invention in detail with respect to some preferred embodiments thereof, the following general definitions are provided. Unless otherwise defined below, terms are used herein as they are generally used in the art to which they pertain.

[0084] The invention will be described with reference to specific embodiments and certain accompanying drawings, but the invention is not limited thereto, but is defined solely by the claims. The use of the term "comprising" in this specification and claims does not exclude other elements. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined below as including at least a certain number of embodiments, it should also be understood as disclosing a group preferably consisting only of these embodiments.

[0085] For the purposes of this invention, the term "obtained" is considered to be a preferred embodiment of the term "available". If, for example, an antibody is defined below as available from a particular source, this should also be understood as disclosing an antibody obtained from that source.

[0086] 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 antigenic epitope bound to an MHC molecule. A TCR may include a TCRα polypeptide chain and a TCRβ polypeptide chain. “Tumor-specific TCR” refers to a TCR that specifically recognizes tumor antigens expressed by tumor cells. “TCR-T” refers to a T cell expressing a recombinant TCR. The “antigen-binding domain” of a TCR refers to a polypeptide that, due to its primary, secondary, or tertiary sequence and / or post-translational modifications and / or charge, binds to an antigen with a high degree of specificity. The antigen-binding domain may be derived from any portion or fragment of the TCR that retains the antigen-binding activity of the TCR as part of the TCR. The antigen-binding portion encompasses, for example, a portion of the TCR that retains specific binding to the amino acid sequence of MAGE-A1 or a fragment thereof, or a portion that, to a similar, equal, or greater degree, the ability to detect, treat, or prevent cancer compared to a wild-type TCR. The TCR antigen-binding domain may comprise, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more of the full-length TCR. The antigen-binding domain may comprise the antigen recognition portion of any one or both of the α and β chains of the TCR, such as portions of the complementarity-determining regions CDR1, CDR2, and CDR3 of the variable regions of the α and / or β chains of the TCR. The antigen-binding domain may comprise the following amino acid sequences: CDR1, CDR2, CDR3 of the α chain, CDR1, CDR2, 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; or the amino acid sequences of CDR1, CDR2, and CDR3 of both the α and β chains. In one embodiment of the invention, the antigen-binding portion may include, for example, a variable region of the TCR, which includes a combination of the aforementioned CDR regions. In this regard, the antigen-binding portion may contain the amino acid sequence of the variable region (Vα) of the α chain of the TCR, the amino acid sequence of the variable region (Vβ) of the β chain, or the amino acid sequence of both Vα and Vβ. In one embodiment of the invention, the antigen-binding portion may contain a combination of a variable region and a constant region. In this regard, the antigen-binding portion may contain the full-length α or β chain of the TCR, or both the α and β chains.

[0087] As used herein, “immune checkpoint inhibitor” refers to agents (including antibodies) that inhibit or block inhibitory immune checkpoint molecules on immune cells (such as T cells) or tumor cells. “Immune checkpoint molecules” include molecules that enhance immune signaling against tumor cells (i.e., “co-stimulatory molecules”) or molecules that reduce immune signaling against tumor cells (i.e., “inhibitory immune checkpoint molecules”).

[0088] As used herein, “treatment” is a means of achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reducing symptoms of another disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the onset or recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing (partial or complete) remission of the disease, reducing the dosage of one or more other medications required to treat the disease, delaying the progression of the disease, increasing quality of life, and / or prolonging survival. “Treatment” also encompasses a reduction in the pathological outcome of a disease (such as cancer). The methods of this invention are contemplated for any or more of these therapeutic aspects.

[0089] As used in this paper, “isolated” constructs (e.g., MAGE-A1 TCR) are (1) not associated with proteins found in nature, (2) do not contain other proteins from the same source, (3) are expressed by cells from different species, or (4) are not found in nature.

[0090] As used herein, the term “isolated nucleic acid” is intended to mean a nucleic acid of genomic, cDNA or synthetic origin or a combination thereof, in which, by virtue of its origin, the “isolated nucleic acid” (1) is not associated with all or part of the polynucleotides of the “isolated nucleic acid” found in nature, (2) is operatively linked to polynucleotides in nature that are not linked to it, or (3) is not present in nature as part of a larger sequence.

[0091] As used herein, “delayed” disease development (such as cancer) means the postponement, inhibition, slowing, delay, stabilization, and / or postponement of disease development. This delay can be of varying lengths, depending on the individual’s medical history and / or current treatment. A sufficient or significant delay, if apparent to a person skilled in the art, may effectively cover prevention, as the individual will not develop the disease. A method of “delayed” disease development (such as cancer), when compared to not using the method, is a method of reducing the likelihood of disease development and / or reducing the severity of the disease in a given period. This simulation is generally based on clinical studies using statistically significant numbers of individuals. Cancer development can be detected using standard methods, including but not limited to computed tomography (CAT) scans, magnetic resonance imaging, abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable, and includes occurrence, recurrence, and onset.

[0092] The terms “individual,” “subject,” and “patient” are used interchangeably herein to describe mammals, including humans. Individuals include, but are not limited to, humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human. In some embodiments, the individual suffers from a disease such as cancer. In some embodiments, the individual requires treatment.

[0093] As understood in the art, an "effective amount" refers to an amount of composition (e.g., engineered immune cells expressing TCRs) sufficient to produce the desired therapeutic outcome (e.g., reducing the severity or duration of one or more cancer symptoms, stabilizing the severity of one or more cancer symptoms, or eliminating one or more cancer symptoms). For therapeutic use, beneficial or desired outcomes include, for example, reducing symptoms (biochemical, histological, and / or behavioral aspects) caused by one or more diseases, including complications and intermediate pathological phenotypes presented during disease development; increasing the quality of life of patients with the disease; reducing the dosage of other drugs required to treat the disease; enhancing the effect of another drug; delaying disease progression; and / or prolonging patient survival.

[0094] "Adjuvant therapy" refers to a clinical setting in which an individual has a history of cancer and has generally (but not necessarily) responded to treatments, including but not limited to surgery (e.g., surgical resection), radiation therapy, and chemotherapy. However, due to their cancer history, these individuals are considered to be at risk of disease progression. The treatment or administration in "adjuvant therapy" refers to the subsequent treatment modality. The level of risk (e.g., when an individual in adjuvant therapy is considered "high-risk" or "low-risk") depends on several factors, most commonly the degree of disease at the time of initial treatment.

[0095] "Neoadjuvant therapy" refers to a clinical setting in which the approach is taken prior to the primary / decisive therapy.

[0096] As used herein, “combination therapy” means that the first agent is administered in combination with another agent. “In combination with” means that a form of treatment is administered in addition to another form of treatment, such as the combinations described herein (e.g., engineered immune cells expressing TCRs) administered to the same individual in addition to the administration of another agent (such as an immune checkpoint inhibitor). Therefore, “in combination with” means that a form of treatment is administered before, during, or after the delivery of another form of treatment to the individual. Such combinations are considered part of a single treatment regimen.

[0097] As used herein, the term "simultaneous administration" means that the first and second therapies in combination therapy are administered at intervals not exceeding about 15 minutes (such as not exceeding about 10, 5, or 1 minute). When the first and second therapies are administered simultaneously, they may be contained in the same composition (e.g., a composition comprising both the first and second therapies) or in different compositions (e.g., the first therapy is in one composition and the second therapy is contained in another composition).

[0098] As used herein, the term "sequential administration" means that the first and second therapies in a combination therapy are administered at intervals of more than about 15 minutes (such as more than about 20, 30, 40, 50, 60, or more minutes). The first or second therapy may be administered first. The first and second therapies may be contained in different compositions, which may be contained in the same or different packages or kits.

[0099] As used in this article, the term "parallel administration" means that the administration of the first therapy and the administration of the second therapy in a combination therapy overlap.

[0100] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, for example, that can be incorporated into a pharmaceutical composition administered to an individual without causing any significant undesirable biological effects or interacting in a harmful manner with any other component in the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required toxicology and manufacturing testing standards and / or are included in the inactive ingredient guidelines prepared by the U.S. Food and Drug Administration.

[0101] The following definitions can be used to assess response based on target lesions: “Complete response” or “CR” means that all target lesions have disappeared; “Partial response” or “PR” means that the sum of the longest diameters of the target lesions has decreased by at least 30%, with reference to baseline SLD; “Stable disease” or “SD” means that the shrinkage of the target lesions is insufficient to meet the criteria for PR, and its increase is insufficient to meet the criteria for PD, with reference to the lowest SLD since the start of treatment; and “Progressive disease” or “PD” means that the SLD of the target lesions has increased by at least 20% (with reference to the lowest SLD recorded since the start of treatment), or that one or more new lesions have appeared.

[0102] The following definitions of response assessment can be used to assess non-target lesions: “Complete response” or “CR” means that all non-target lesions have disappeared; “stable disease” or “SD” means that one or more non-target lesions that do not meet the criteria for CR or PD persist; and “progressive disease” or “PD” means “clear progression” of existing (multiple) non-target lesions, or the appearance of one or more new lesions is considered as disease progression (if an individual’s PD is assessed at a point in time based entirely on the progression of (multiple) non-target lesions, additional criteria must be met).

[0103] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. It is understood that due to human or accidental mutations, the DNA content of all progeny may not be entirely identical. Variant progeny with the same function or biological activity as the original cell are included.

[0104] As used herein, the terms “specifically bind to,” “recognize,” “specifically recognize,” “target,” or “specific to” refer to measurable and reproducible interactions, 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, which determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a TCR that binds to or specifically binds to a target epitope is a TCR that binds to the target epitope / MHC complex with greater affinity, binding strength, ease, and / or a longer duration (compared to its binding to other epitope / MHC complexes). In one embodiment, as measured (e.g., by radioimmunoassay (RIA)), the degree of binding of a TCR to an unrelated epitope / MHC complex is less than about 10% of the binding of the TCR to the target epitope / MHC complex. In some embodiments, the TCR that specifically binds to the target epitope (i.e., the target epitope / MHC complex) has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In some embodiments, the TCR specifically binds to an epitope on a conserved protein from different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

[0105] As used herein, the term “isolated nucleic acid” is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which, due to its origin, (1) is not associated with all or part of the polynucleotides of “isolated nucleic acids” found in nature, (2) is operatively linked to polynucleotides that would not be linked in nature, or (3) is not present in nature as a part of a larger sequence.

[0106] It is understood that the aspects and embodiments of the present invention described herein include aspects and embodiments that are “consisting” and / or “substantially constituted”.

[0107] The term "about" as used in this document includes (and describes) variations of that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0108] The phrase “about X to Y” used in this article has the same meaning as “about X to about Y”.

[0109] As used in this article, referring to "not / not" as a value or parameter generally means and describes a value or parameter "other than". For example, "this method is not used to treat type X cancer" means that this method is used to treat types of cancer other than X.

[0110] As used herein and in the appended claims, the singular forms “a” and “the” both include the plural designation, unless the context clearly indicates otherwise.

[0111] II. MAGE-A1 tumor-specific TCR

[0112] This application provides a tumor-specific TCR that specifically recognizes tumor MAGE-A1 and / or specifically recognizes the MHC / MAGE-A1 epitope complex. In some embodiments, the tumor-specific TCR specifically recognizes MAGE-A1. In some embodiments, the tumor-specific TCR specifically recognizes the MHC / MAGE-A1 epitope complex. Nucleic acids and vectors encoding tumor-specific TCRs, and engineered immune cells expressing tumor-specific TCRs are also within the scope of this patent application.

[0113] Exemplary TCRs identified using the methods described herein are shown in Table 1 below. The V, J, and C segments are named according to the IMGT database. Other nomenclature and delineation algorithms known in the art may be used. For example, according to IMGT, the TCR chain includes: FR1 from amino acid positions 1 to 26, CDR1 from amino acid positions 27 to 38, FR2 from amino acid positions 39 to 55, CDR2 from amino acid positions 56 to 65, FR3 from amino acid positions 66 to 104, and CDR3 from amino acid positions 105 to 117 (for rearranged VJ and VDJ genes), and FR4 from amino acid positions 118 to 129. See, for example, Lefranc, M.-P., The Immunologist, 7, 132-136 (1999), and world wideweb.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html. The CDR1, CDR2, and CDR3 of the exemplary TCR are shown in the "Sequence List" section.

[0114] Table 1: Exemplary Tumor-Specific TCRs

[0115]

[0116]

[0117] In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, which specifically binds to an epitope of the MAGE-A1 peptide, such as a MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:365-369. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain specifically binds to an MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:365. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain specifically binds to an MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:366. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain specifically binds to an MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:367. In some embodiments, the MAGE-A1 tumor-specific TCR specifically binds to an MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:368. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain specifically binds to a MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:369. In some embodiments, a MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:365-396 is provided. In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided that specifically binds to a MAGE-A1 peptide / MHC complex, wherein MAGE-A1 comprises a MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:365-369. In some embodiments, the MHC protein in the MAGE-A1 peptide / MHC complex is an MHC class II protein. In some embodiments, the MHC class II protein is HLA-DRA. In some embodiments, the MHC class II protein is HLA-DRB. In some embodiments, HLA-DRA is DRA*01:01. In some embodiments, HLA-DRB is DRB1*07:01. In some embodiments, HLA-DRB is DRB1*12:02. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCRs or their antigen-binding domains is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 257-287Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 265-279 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex.

[0118] In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising the amino acid sequence of any one of SEQ ID NOs: 3, 6, 17, 20, 31, 34, 45, 48, 59, 62, 73, 76, 87, 90, 101, 104, 115, 118, 129, 132, 143, 146, 157, 160, 171, 174, 185, 188, 199, and 202. In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising: (a) TCRαCDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:3; and TCRβCDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:6; (b) TCRαCDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:17; and TCRβCDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:20; (c) TCRαCDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:31; and TCRβ ... NO:34 has at least about 90% sequence identity (e.g., 100% identity); (d) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:45; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:48; (e) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:59; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:62; (f) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:73; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:73; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:45; NO:76 has at least about 90% sequence identity (e.g., 100% identity); (g) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:87; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:90; (h) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:101;And TCRβCDR3, which has at least about 90% sequence identity with SEQ ID NO:104 (e.g., 100% identity); (i) TCRαCDR3, which has at least about 90% sequence identity with SEQ ID NO:115 (e.g., 100% identity); and TCRβCDR3, which has at least about 90% sequence identity with SEQ ID NO:118 (e.g., 100% identity); (j) TCRαCDR3, which has at least about 90% sequence identity with SEQ ID NO:129 (e.g., 100% identity); and TCRβCDR3, which has at least about 90% sequence identity with SEQ ID NO:132 (e.g., 100% identity); (k) TCRαCDR3, which has at least about 90% sequence identity with SEQ ID NO:143 (e.g., 100% identity); and TCRβCDR3, which has at least about 90% sequence identity with SEQ ID NO:143 (e.g., 100% identity); and TCRβCDR3, which has at least about 90% sequence identity with SEQ ID NO:104 (e.g., 100% identity); NO:146 has at least about 90% sequence identity (e.g., 100% identity); (l) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:157; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:160; (m) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:171; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:174; (n) TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:185; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:185; and TCRβCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:157; NO:188 has at least about 90% sequence identity (e.g., 100% identity); or (o)TCRαCDR3, which has at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO:199;And TCRβCDR3, which has at least about 90% sequence identity with SEQ ID NO:202 (e.g., 100% identity). In some embodiments, the antigen-binding construct of any of (a) to (c) specifically binds to the epitope of the MAGE-A1 peptide comprising the amino acid sequence of SEQ ID NO:365 and / or 366. In some embodiments, the antigen-binding construct of any of (d) to (f) specifically binds to the epitope of the MAGE-A1 peptide comprising the amino acid sequence of NO:365 and / or 367. In some embodiments, the antigen-binding construct of any of (g) to (i) specifically binds to the MAGE-A1 epitope comprising the amino acid sequence of any of NO:365-368. In some embodiments, the antigen-binding construct of (h) to (j) specifically binds to the MAGE-A1 epitope comprising the amino acid sequence of SEQ ID NO:365 and / or 369. In some embodiments, the antigen-binding construct of any of (m) to (o) specifically binds to the epitope of the MAGE-A1 peptide, including the amino acid sequence NO: 365 and / or 367.

[0119] In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising a TCRα chain comprising (a) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 9, 23, or 37; (b) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 51, 65, or 79; (c) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 93, 107, or 121; (d) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 135, 149, or 163; or (e) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 177 or 191.

[0120] In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising a TCRβ chain comprising (a) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 12, 26, or 40; (b) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 54, 68, or 82; (c) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 96, 110, or 124; (d) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 138, 152, or 166; or (e) CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 180 or 194.

[0121] In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising: (a) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 9, 23, or 37; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 12, 26, or 40; (b) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 51, 65, or 79; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 54, 68, or 82; (c) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 93, 107, or 121; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 96, 110, or 124; and (d) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 93, 107, or 121; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 96, 110, or 124; CDR of any of the amino acid sequences NO: 135, 149, or 163; and TCRβ chain, including CDR of any of the amino acid sequences SEQ ID NO: 138, 152, or 166; or (e) TCRα chain, including CDR of any of the amino acid sequences SEQ ID NO: 177 or 191; and TCRβ chain, including CDR of any of the amino acid sequences SEQ ID NO: 180 or 194.

[0122] The tumor-specific TCR or its antigen-binding domain described herein also includes a TCR constant domain. In some embodiments, the tumor-specific TCR or its antigen-binding domain comprises: a TCRα chain comprising the TCRα constant domain (TRAC) of any one of SEQ ID NO: 9, 23, 37, 51, 65, 79, 93, 107, 121, 135, 149, 163, 177, and 191, or a variant thereof; and a TCRβ chain comprising the TCRβ constant domain (TRBC) of any one of the amino acid sequences of SEQ ID NO: 12, 26, 40, 54, 68, 82, 96, 110, 124, 138, 152, 166, 180, and 194, or a variant thereof. In some embodiments, the tumor-specific TCR or its antigen-binding domain comprises: human TRAC and human TRBC, such as human Cα and human Cβ1, or human Cα and human Cβ2. In some implementations, the tumor-specific TCR or its antigen-binding domain includes murine TRAC and murine TRBC, such as murine Cα and murine Cβ1, or murine Cα and murine Cβ2.

[0123] In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain is a human TCR. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain is a chimeric TCR, such as a murine TCR, for example, a TCR comprising murine constant regions of the TCR α and β chains. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain comprises a human TCR variable region and a TCR constant region derived from a non-human species (such as a mouse).

[0124] In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising: (a) a TCRα chain comprising an amino acid sequence of any one of SEQ ID NO: 9, 23, and 37 having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity); and a TCRβ chain comprising an amino acid sequence of any one of SEQ ID NO: 12, 26, and 40 having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity); and (b) a TCRα chain comprising an amino acid sequence of SEQ ID NO: 9, 23, and 37 having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity); (c) A TCRβ chain comprising an amino acid sequence ... (d) A TCRα chain comprising an amino acid sequence ... The amino acid sequences of NO:177 and 191 have at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity).and the TCRβ chain, comprising an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity) of any of the amino acid sequences of SEQ ID NO: 180 and 194.

[0125] Also provided is an isolated nucleic acid encoding the TCRα chain and / or the TCRβ chain of a tumor-specific TCR or its antigen-binding domain according to any of the above-mentioned tumor-specific TCRs or their antigen-binding domains; and a vector comprising the isolated nucleic acid. 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.

[0126] III. Immune cells expressing MAGE-A1 tumor-specific TCRs or their antigen-binding domains

[0127] One aspect of this patent application provides an engineered immune cell comprising a tumor-specific TCR or antigen-binding domain thereof based on any one of the tumor-specific TCRs or antigen-binding domains thereof, an isolated nucleic acid, or a vector.

[0128] This application provides engineered immune cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, an effector cell (e.g., a T cell) is provided that presents any of the MAGE-A1 tumor-specific TCRs or their antigen-binding domains as described herein on its surface. In some embodiments, the effector cell comprises nucleic acid encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain, wherein the MAGE-A1 tumor-specific TCR or its antigen-binding domain is expressed from the nucleic acid and located on the effector cell surface. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain is exogenously expressed and combined with the effector cell. 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.

[0129] Cellular modifications used to disrupt 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), etc. For example, in some embodiments, an effector cell (e.g., a T cell) is provided comprising a nucleic acid encoding any of the MAGE-A1 tumor-specific TCR or its antigen-binding domain as described herein, wherein the MAGE-A1 tumor-specific TCR or its antigen-binding domain is expressed from the nucleic acid and localized on the effector cell surface. In some embodiments, an effector cell (e.g., a T cell) is provided comprising a first nucleic acid sequence encoding a first polypeptide chain encoding a first polypeptide chain encoding a first polypeptide chain encoding a second ... 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 the MAGE-A1 tumor-specific TCR or its antigen-binding domain is under promoter control. 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 times (e.g., at least about 2, 3, 4, 5, or greater) times the expression of the second polypeptide chain. In some embodiments, the expression of the first polypeptide chain does not exceed about half of the expression of the second polypeptide chain (e.g., not more than about 1 / 2, 1 / 3, 1 / 4, 1 / 5 or less).

[0130] The expression of MAGE-1 tumor-specific TCR or its antigen-binding domain can be determined at the mRNA or protein level. mRNA expression levels can be measured using various well-known methods to determine the amount of mRNA transcribed from nucleic acids, including Northern blotting, quantitative RT-PCR, and microarray analysis. Protein expression levels can be measured using known methods, including immunocytochemical staining, enzyme-linked immunosorbent assay (ELISA), Western blotting, luminescence analysis, mass spectrometry, high-performance liquid chromatography (HPLC), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0131] In some embodiments, an effector cell (e.g., a T cell) of a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, which expresses on its surface any of the MAGE-A1 tumor-specific TCR or its antigen-binding domain as described herein, wherein the effector cell of the MAGE-A1 tumor-specific TCR or its antigen-binding domain comprises a mammalian expression vector and a viral vector (e.g., a vector derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses) integrated with the host genome, which comprises a first nucleic acid sequence encoding a first polypeptide chain encoding a first polypeptide chain of the MAGE-A1 tumor-specific TCR or its antigen-binding domain and a second nucleic acid sequence encoding a second polypeptide chain of the MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, a promoter operatively attached to the 5' end of a first nucleic acid sequence is present, and a nucleic acid adapter selected from internal ribosome entry sites (IRES) and nucleic acids encoding self-cleaving 2A peptides (e.g., P2A, T2A, E2A, or F2A) is present to link the 3' end of the first nucleic acid sequence to the 5' end of a second nucleic acid sequence, wherein the first and second nucleic acid sequences are transcribed into a single RNA under the control of the promoter. In some embodiments, a promoter operatively attached to the 5' end of a second nucleic acid sequence is present, and a nucleic acid adapter selected from internal ribosome entry sites (IRES) and nucleic acids encoding self-cleaving 2A peptides (e.g., P2A, T2A, E2A, or F2A) is present to link the 3' end of the second nucleic acid sequence to the 5' end of the first nucleic acid sequence, wherein the first and second nucleic acid sequences 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 implementations, effector cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

[0132] In some embodiments, an effector cell (e.g., a T cell) expressing MAGE-A1 tumor-specific TCR on its surface is provided, comprising a) a first nucleic acid sequence encoding a first polypeptide chain, the 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, the 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, the 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, the 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, the 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, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:40; and / or g) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:51 and h) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:54; and / or i) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:12. The amino acid sequence of SEQ ID NO:65 and j) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:68; and / or k) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:79 and l) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:82; and / or m) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:93 and n) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:96; and / or o) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:107 and p) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:110; and / or q) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:121 and r) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:68. The amino acid sequence of NO:124; and / or s) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:135 and t) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:138;And / or u) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 149 and v) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 152; and / or w) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 163 and x) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 166; and / or y) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 177 and z) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 180; and / or aa) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 191 and ab) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 194. In some embodiments, effector cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain further comprise a P2A nucleic acid sequence encoding SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 111, 125, 139, 153, 167, 181, or 195. In some embodiments, an effector cell (e.g., a T cell) expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain on its surface is provided, comprising a nucleic acid sequence encoding one or more of SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182, and 196.

[0133] In some embodiments, the target antigen of effector cells (e.g., T cells) that express the MAGE-A1 tumor-specific TCR or its antigen-binding domain on the surface is the MAGE-A1 peptide / MHC complex. In some embodiments, the MHC protein in the MAGE-A1 peptide / MHC complex is an MHC-II protein. In some embodiments, the MHC-II protein is HLA-DRA. In some embodiments, the MHC-II protein is HLA-DRB. In some embodiments, HLA-DRA is DRA*01:01. In some embodiments, HLA-DRB is DRB1*07:01. In some embodiments, HLA-DRB is DRB1*12:02. In some embodiments, the effector cell is γδT cell. In some embodiments, the effector cell is αβT 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 implementations, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCRs or their antigen-binding domains is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 265-279 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex.

[0134] In some embodiments, the immune cell is a T cell. In some embodiments, a pharmaceutical composition is provided comprising an engineered immune cell according to any of the engineered immune cells described above, and a pharmaceutically acceptable carrier.

[0135] IV. Preparation of effector cells expressing MAGE-A1 TCR or its antigen-binding domain

[0136] In some embodiments, in one aspect, the present invention provides effector cells (e.g., lymphocytes, such as T cells) expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain. This document provides exemplary methods for preparing effector cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain (e.g., MAGE-A1 tumor-specific TCR effector cells, such as MAGE-A1 tumor-specific TCRT cells). In some embodiments, MAGE-A1 tumor-specific TCR effector cells (e.g., MAGE-A1 tumor-specific TCRT cells) can be generated by introducing a nucleic acid (including, for example, a lentiviral vector) encoding one or more MAGE-A1 tumor-specific TCRs or their antigen-binding domains (e.g., any of the MAGE-A1 tumor-specific TCRs or their antigen-binding domains described herein) that specifically bind to a target antigen (e.g., a disease-related antigen) into the effector cells. The introduction of one or more nucleic acids into the effector cells can be achieved using techniques known in the art, such as those described herein with respect to nucleic acids. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells of the present invention (e.g., MAGE-A1 tumor-specific TCRT cells) are capable of replicating in vivo, producing long-term durability that can contribute to sustained control of diseases (e.g., cancer) associated with the expression of target antigens.

[0137] In some embodiments, the present invention relates to the treatment of patients with or at risk of developing a disease and / or condition associated with the expression of a target antigen (also referred to herein as a "target antigen-positive" or "TA-positive" disease or condition), including, for example, cancer, by administering genetically modified T cells via lymphocyte infusion. The genetically modified T cells express any of the MAGE-A1 tumor-specific TCR or its antigen-binding domain as described herein. In some embodiments, autologous lymphocyte infusion is used in the treatment. Autologous PBMCs are collected from the patient requiring treatment and the 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 expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen is provided (also referred to herein as a "MAGE-A1 tumor-specific TCRT cell"). The MAGE-A1 tumor-specific TCRT cells of the present invention can undergo robust in vivo T cell expansion and establish target antigen-specific memory cells that persist in high concentrations in the blood and bone marrow for extended periods. In some embodiments, the MAGE-A1 tumor-specific TCRT cells of the present invention, infused into a patient, can eliminate target antigen-presenting cells, such as target antigen-presenting cancer cells, in vivo from patients with target antigen-related diseases. In some embodiments, the MAGE-A1 tumor-specific TCRT cells of the present invention, infused into a patient, can eliminate target antigen-presenting cells, such as target antigen-presenting cancer cells, in vivo from patients with target antigen-related diseases that are difficult to treat with at least one known therapy.

[0138] Prior to T-cell expansion and genetic modification, T-cells are derived from an individual. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T-cell lines available in the art can be used. In some embodiments of the invention, T cells can be obtained using any number of techniques known to those skilled in the art (e.g., Ficoll). TMCells are isolated from blood units collected from an individual. In some embodiments, cells from an individual's circulating blood are obtained via apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, the cells collected via apheresis may be washed to remove the plasma fraction and the cells placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. As will be readily apparent to those skilled in the art, the washing step can be performed by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifugation (e.g., the Cobe 2991 cell processor Baxter CytoMate or the Haemonetics cell preserver 5) according to the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyteA, or other physiological saline solutions with or without buffers. Alternatively, unwanted components of the hemocytocyte separation sample can be removed and the cells can be directly resuspended in the culture medium.

[0139] In some implementations, this is achieved by lysing red blood cells and, for example, by percolation. TM T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugation to deplete monocytes. Specific subsets of T cells (e.g., CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T5 cells) can be further isolated using positive or negative selection techniques. For example, in some embodiments, T cells are isolated by beads that bind to anti-CD3 / anti-CD28 (i.e., 3×28) (e.g., [missing information]). Incubation of T cells with M-450CD3 / CD28T for a period sufficient for positive selection of desired T cells isolating T cells. In some embodiments, the incubation period is approximately 30 minutes. In some embodiments, the incubation period ranges from 30 minutes to 36 hours or longer (inclusive of all values ​​between these). In some embodiments, the incubation period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the incubation period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. Regarding the isolation of T cells from patients with leukemia, using a longer incubation time (e.g., 24 hours) can increase cell yield. A longer incubation time can be used to isolate T cells in any situation where fewer T cells are present compared to other cell types, such as the isolation of tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, using a longer incubation time can improve the capture efficiency of CD8+ T cells. Therefore, T cell subsets can be preferentially selected for or for other time points at the start of culture or during the process by simply shortening or lengthening the time allowed for T cells to bind to CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T cell ratio. Additionally, T cell subsets can be preferentially selected for or for other desired time points at the start of culture by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces. Those skilled in the art will recognize that multiple selection rounds may also be used in the context of this invention. In some embodiments, a selection procedure may be required and “unselected” cells may be used during activation and expansion. “Unselected” cells may also be subject to other selection rounds.

[0140] Enriching a T cell population through negative selection can be achieved using a combination of antibodies targeting surface markers specific to the negatively selected cells. One approach involves sorting and / or selecting cells via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells through negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, it may be necessary to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some embodiments, T regulatory cells are depleted using anti-CD25 binding beads or other similar selection methods.

[0141] To separate the desired cell population through positive or negative selection, cell concentration and surface area (e.g., particles, such as beads) can be varied. In some embodiments, it may be necessary to significantly reduce the volume of beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in some embodiments, a concentration of about 2 billion cells / mL is used. In some embodiments, a concentration of about 1 billion cells / mL is used. In some embodiments, a concentration greater than about 100,000,000 cells / mL is used. In some embodiments, a cell concentration of any one 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 / mL is used. In some embodiments, cell concentrations of approximately 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, or 100,000,000 cells / mL are used. In some embodiments, concentrations of approximately 125,000,000 or approximately 150,000,000 cells / mL are used. Using high concentrations can lead to increased cell yield, cell activation, and cell proliferation. Furthermore, using high cell concentrations allows for more effective capture of cells that may weakly express the target antigen of interest (e.g., CD28-negative T cells) or samples containing a large number of tumor cells (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may be therapeutically valuable and desirable. For example, using high cell concentrations allows for more effective selection of CD8+ T cells that typically have weak CD28 expression.

[0142] In some embodiments of the invention, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that, following certain cancer treatments, particularly those using drugs that disrupt the immune system, the quality of the T cells obtained shortly after treatment, during the period when the patient typically recovers from treatment, is optimal, or their ability to expand in vitro is improved. Similarly, enhanced transplantation and in vivo expansion of these cells after in vitro manipulation using the methods described herein are preferred. Therefore, the context of the 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, conditions can be established within the individual using migration (e.g., migration with GM-CSF) and conditioning protocols, particularly during a defined time window after treatment, to reproliferate, recirculate, regenerate, and / or expand specific cell types. 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 the desired MAGE-A1 tumor-specific TCR or its antigen-binding domain, T cells can generally be activated and expanded using methods described, for example, in the following: U.S. Patent Nos. 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; and U.S. Patent Application Publication No. 20060121005.

[0143] Generally, the T cells of the present invention proliferate by contacting a surface to which an agent stimulating CD3 / TCR complex-related signals and a ligand stimulating co-stimulatory molecules on the T cell surface are attached. Specifically, the T cell population can be stimulated, for example, by contacting an anti-CD3 antibody or its antigen-binding domain, or an anti-CD2 antibody immobilized on the surface, or by contacting a protein kinase C activator (e.g., bryostatin) bound to a calcium ionocarrier. Ligands binding to helper molecules on the T cell surface are used to co-stimulate them. For example, the T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Anti-CD3 and anti-CD28 antibodies are used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (DiaClone, ...). France), can be used as other methods generally known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0144] Gene modification

[0145] In some embodiments, the MAGE-A1 tumor-specific TCR effector cells of the present invention (e.g., MAGE-A1 tumor-specific TCRT cells) are generated by transducing effector cells (e.g., T cells prepared by the methods described herein) with a viral vector encoding the MAGE-A1 tumor-specific TCR as described herein. The viral vector delivery system includes DNA and RNA viruses that have a free or integrated genome after delivery to the effector cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel and Feigner, TIBTECH 11:211-217 (1993); Mitani and Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 557:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); and Yu et al., Gene Therapy 1:13-26 (1994). In some embodiments, the viral vector is a lentiviral vector, and the MAGE-A1 tumor-specific TCR effector cells comprise a lentiviral vector integrated into the genome of the MAGE-A1 tumor-specific TCR effector cells. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are MAGE-A1 tumor-specific TCRT cells comprising a lentiviral vector integrated into their genome. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells modified to block or reduce the expression of one or both of the endogenous TCR chains. For example, in some embodiments, the MAGE-A1 tumor-specific TCR effector cells are αβT cells modified to block or reduce the expression of TCR α and / or β chains, or γδT cells modified to block or reduce the expression of TCR γ and / or δ chains. Cellular modifications used to disrupt gene expression include any such techniques known in the art, including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., gene knockout based on CRISPR or TALEN), etc.

[0146] In some implementations, MAGE-A1 tumor-specific TCRT cells with reduced expression of one or both of the endogenous TCR chains of T cells are generated using a CRISPR / Cas system. For reviews of gene-editing CRISPR / Cas systems, see, for example, Jian W and Marraffini LA, Annu. Rev. Microbiol. 69, 2015; Hsu PD et al., Cell, 157(6):1262-1278, 2014; and O'Connell MR et al., Nature 516:263-266, 2014. In some implementations, MAGE-A1 tumor-specific TCRT cells with reduced expression of one or both of the endogenous TCR chains of T cells are generated using TALEN-based genome editing.

[0147] Enrichment

[0148] In some embodiments, a method is provided for enriching MAGE-A1 tumor-specific TCR effector cells from any of the MAGE-A1 tumor-specific TCR effector cells described herein within a heterogeneous cell population. A specific subset of MAGE-A1 tumor-specific TCR effector cells (e.g., MAGE-A1 tumor-specific TCRT cells) that specifically bind to a target antigen can be enriched using a positive selection technique. For example, in some embodiments, MAGE-A1 tumor-specific TCR effector cells (e.g., T cells expressing MAGE-A1 tumor-specific TCR) are enriched by incubating with beads bound to the target antigen for a period sufficient to positively select the desired MAGE-A1 tumor-specific TCR effector cells. In some embodiments, the period is about 30 minutes. In some embodiments, the period is in the range of 30 minutes to 36 hours or longer (inclusive of all values ​​between these). In some embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For the isolation of MAGE-A1 tumor-specific TCR effector cells present in low concentrations from heterogeneous cell populations, using a longer incubation time (e.g., 24 hours) can increase cell yield. A longer incubation time can be used to isolate MAGE-A1 tumor-specific TCR effector cells in any situation where there are fewer MAGE-A1 tumor-specific TCR effector cells compared to other cell types.

[0149] Those skilled in the art will recognize that multiple selection wheels may also be used in the context of this invention. To separate the desired MAGE-A1 tumor-specific TCR effector cell population by positive or negative selection, cell concentration and surface area (e.g., particles, such as beads) can be varied. In some embodiments, it may be necessary to significantly reduce the volume of beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in some embodiments, a concentration of approximately 2 billion cells / mL is used. In some embodiments, a concentration of approximately 1 billion cells / mL is used. In some embodiments, a concentration greater than approximately 100 million cells / mL is used. In some embodiments, a cell concentration of approximately 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 / mL is used. In some embodiments, a cell concentration of approximately 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 approximately 125,000,000 or approximately 150,000,000 cells / mL is used. Using high concentrations can lead to increased cell yield, cell activation, and cell proliferation. Furthermore, using high cell concentrations allows for more effective capture of MAGE-A1 tumor-specific TCR effector cells that may weakly express the MAGE-A1 tumor-specific TCR or its antigen-binding domain.

[0150] In some of the embodiments described herein, enrichment results in minimal or substantially no depletion of MAGE-A1 tumor-specific TCR effector cells. For example, in some embodiments, enrichment results in depletion of less than about 50% (e.g., less than any one of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of MAGE-A1 tumor-specific TCR effector cells. Effector cell depletion can be determined by any means known in the art, including any of those described herein. In some of the embodiments described herein, enrichment results in minimal or substantially no final differentiation of MAGE-A1 tumor-specific TCR effector cells. For example, in some embodiments, enrichment results in final differentiation of less than about 50% (e.g., less than any one of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of MAGE-A1 tumor-specific TCR effector cells. Effector cell differentiation can be determined by any means known in the art, including any of those described herein.

[0151] In some of the embodiments described herein, enrichment results in minimal or substantially no internalization of the MAGE-A1 tumor-specific TCR or its antigen-binding domain on MAGE-A1 tumor-specific TCR effector cells. For example, in some embodiments, enrichment results in less than about 50% (e.g., less than any one of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the MAGE-A1 tumor-specific TCR or its antigen-binding domain on MAGE-A1 tumor-specific TCR effector cells. Internalization of the MAGE-A1 tumor-specific TCR or its antigen-binding domain on MAGE-A1 tumor-specific TCR effector cells can be determined by any means known in the art, including any of those described herein.

[0152] In some of the embodiments described herein, enrichment leads to an increase in the proliferation of MAGE-A1 tumor-specific TCR effector cells. For example, in some embodiments, enrichment leads to an increase in the number of MAGE-A1 tumor-specific TCR effector cells by at least about 10% after enrichment (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000% or more). Therefore, in some embodiments, a method is provided for enriching MAGE-A1 tumor-specific TCR effector cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain specifically binding to a target antigen from a heterogeneous cell population, comprising: a) contacting the heterogeneous cell population with a ligand containing the target antigen or one or more epitopes contained therein to form a complex containing MAGE-A1 tumor-specific TCR effector cells bound to the ligand; and b) separating the complex from the heterogeneous cell population, thereby producing a cell population enriched with MAGE-A1 tumor-specific TCR effector cells. In some embodiments, the ligand is immobilized to a solid carrier. In some embodiments, the solid carrier is a microparticle (e.g., beads). In some embodiments, the solid carrier is a surface (e.g., the bottom of a pore). In some embodiments, the ligand is tagged. In some embodiments, the tag is a fluorescent molecule, an affinity tag, or a magnetic tag. In some embodiments, the method further comprises eluting the MAGE-A1 tumor-specific TCR effector cells from the ligand and recovering the eluent.

[0153] In some implementations, T cells can be enriched by screening for CD3 and IFN-γ using a FACS method. In some implementations, T cells can be enriched by screening for IFN-γ using an ELISA method.

[0154] Document filtering

[0155] To isolate candidate MAGE-A1 tumor-specific TCRs or antigen-binding domain constructs thereof that are specific to a target antigen, a library of MAGE-A1 tumor-specific TCRs or their antigen-binding domains (e.g., cells expressing a nucleic acid library encoding multiple MAGE-A1 tumor-specific TCRs or their antigen-binding domains) can be exposed to a ligand containing the target antigen or one or more epitopes 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 support may be a bead surface, a microtiter plate, an immunotube, or any material known in the art suitable for such purposes.

[0156] In some embodiments, the interaction occurs in solution on a labeled ligand target (e.g., a biotin-labeled ligand). In some embodiments, the procedure involves one or more washing steps (panning) for removing nonspecific and non-responsive library members. In some embodiments, the complex is captured by immobilization or by centrifugation to purify the complex in solution. In some embodiments, the affinity member is captured on a soluble biotin-labeled ligand, and then the affinity complex (affinity member and ligand) is immobilized on streptavidin beads. In some embodiments, the solid carrier is a bead. In some embodiments, the beads include, for example, magnetic beads (e.g., from Bangs Laboratories, Polysciencesinc., Dynal Biotech, Miltenyi Biotech, or Quantum Magnetic), nonmagnetic beads (e.g., Pierce and Upstatetechnology), monodisperse beads (e.g., Dynal Biotech and Microparticle GmbH), and polydisperse beads (e.g., Chemagen). The use of magnetic beads has been described in detail in the literature (Uhlen, M et al. (1994) Advances in Biomagnetic Separation, Bio Techniques press, 5 Westborough, MA). In some embodiments, affinity members are purified by positive selection. In some embodiments, affinity members are purified by negative selection to remove undesirable library members. In some embodiments, affinity members are purified by both positive and negative selection steps.

[0157] Generally, the techniques used to prepare library constructs are based on known genetic engineering techniques. In this regard, the nucleic acid sequence encoding the MAGE-A1 tumor-specific TCR or its antigen-binding domain to be expressed in the library is incorporated into an expression vector suitable for the type of expression system to be used. Appropriate 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.

[0158] In some embodiments, a nucleic acid library is provided comprising sequences encoding a plurality of MAGE-A1 tumor-specific TCRs or their antigen-binding domains according to any of the embodiments described herein. In some embodiments, the nucleic acid library comprises a viral vector encoding a plurality of MAGE-A1 tumor-specific TCRs or their antigen-binding domains. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, a method is provided for screening sequences encoding MAGE-A1 tumor-specific TCRs or their antigen-binding domains specific to a target antigen from a nucleic acid library according to any of the embodiments described herein, comprising: a) introducing the nucleic acid library into a plurality of cells such that MAGE-A1 tumor-specific TCRs or their antigen-binding domains are expressed on the surface of the plurality of cells; b) incubating the plurality of cells with a ligand containing a target antigen or one or more epitopes contained therein; c) collecting cells bound to the ligand; and d) isolating sequences encoding MAGE-A1 tumor-specific TCRs or their antigen-binding domains from the cells collected in step c) thereby identifying MAGE-A1 tumor-specific TCRs or their antigen-binding domains specific to the target antigen. In some embodiments, the method further includes one or more washing steps. In some embodiments, the one or more washing steps are performed between steps b) and c). In some embodiments, the multiple cells are multiple 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 cells bound to the ligand comprises eluting cells from the ligand bound to the solid support and collecting the eluent. In some embodiments, the ligand is tagged. In some embodiments, the tag is a fluorescent molecule, an affinity tag, or a magnetic tag. In some embodiments, collecting cells bound to the ligand comprises separating a complex containing the cells and the tagged ligand. In some embodiments, the cells are dissociated from the complex.

[0159] MHC proteins

[0160] MHC class II proteins are one of two major classes of major histocompatibility complex (MHC) molecules (the other being MHC class I) and are found on almost every nucleated cell in the body. Their function is to present intracellular protein fragments to T cells; healthy cells ignore them, while cells containing foreign or mutated proteins are attacked by the immune system. Antigens presented by class II peptides originate from extracellular proteins; therefore, the MHC class II-dependent antigen presentation pathway is called endocytosis or the exogenous pathway. Loading of MHC class II molecules occurs through phagocytosis; extracellular proteins are endocytosed, digested in lysosomes, and the resulting epitope peptide fragments are loaded onto MHC class II molecules, which then migrate to the cell surface.

[0161] MHC class II molecules are heterodimers composed of two homogeneous peptides: α and β chains. Sub-names such as α1 and α2 refer to individual domains within HLA genes; each domain is typically encoded by a different exon within the gene, and some genes also have other domains encoding leader sequences, transmembrane sequences, etc. Because the antigen-binding grooves of MHC class II molecules are open at both ends, while the corresponding grooves on class I molecules are closed at both ends, antigens presented by MHC class II molecules are longer, generally between 15 and 24 amino acid residues in length.

[0162] The human leukocyte antigen (HLA) gene is the human type of the MHC gene. The three main MHC class II proteins involved in antigen presentation in the human body are HLA-DP, HLA-DQ, and HLA-DR, while other MHC class II proteins, HLA-DM and HLA-DO, are involved in internal antigen processing and loading. In some embodiments, a complex of a peptide of the relevant antigen (e.g., a tumor-associated or virus-encoded antigen) and an MHC class II protein, wherein the MHC class II protein is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, the MHC class II protein is HLA-DP. In some embodiments, the MHC class II protein is HLA-DQ. In some embodiments, the MHC class II protein is HLA-DR. Peptides suitable for generating antigen-binding modules can be determined, for example, based on the presence of HLA (e.g., HLA-DRA*01:01) binding motifs and cleavage sites on the proteasome and immunoproteasome, using computer prediction models known to those skilled in the art. Regarding the prediction of MHC binding sites, such models include (but are not limited to) ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237, 2001) and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. Immunoinformatics Methods in Molecular Biology, Vol. 409(1):75-93, 2007).

[0163] Once a suitable peptide is identified, its synthesis can be performed according to methods well known to those skilled in the art. The peptides of the present invention, due to their relatively small size, can be synthesized directly in solution or on a solid support using known peptide synthesis techniques. Various automated synthesizers are commercially available and can be used according to known methods. Synthesizing peptides in the solution phase has become a recognized procedure for the large-scale production of synthetic peptides, and is therefore a suitable alternative for the preparation of the peptides of the present invention (see, for example, Solid Phase Peptide Synthesis, John Morrow Stewart and Martin et al. Application of Almez-mediated Amidation Reactions to Solution Phase Peptide Synthesis, Tetrahedron Letters, Vol. 39, pp. 1517-1520, 1998).

[0164] In some embodiments, the MHC protein in the MAGE-A1 peptide / MHC complex is an MHC-II protein. In some embodiments, the MHC-II protein is HLA-DRA. In some embodiments, the MHC-II protein is HLA-DRB. In some embodiments, the MHC-II protein is HLA-DPA. In some embodiments, the MHC-II protein is HLA-DPB. In some embodiments, the MHC-II protein is HLA-DQA. In some embodiments, the MHC-II protein is HLA-DQB. In some embodiments, HLA-DRA is DRA*01:01. In some embodiments, HLA-DRB is DRB1*07:01. In some embodiments, HLA-DRB is DRB1*12:02. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCRs or their antigen-binding domains is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 265-279 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex.

[0165] Mutants

[0166] In some embodiments, amino acid sequence variants of the MAGE-A1 TCR or its antigen-binding domain provided herein are included. For example, it may be necessary to improve the binding affinity and / or other biological properties of the MAGE-A1 TCR or its antigen-binding domain. Amino acid sequence variants of the MAGE-A1 TCR or its antigen-binding domain can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the MAGE-A1 TCR or its antigen-binding domain, 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 performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. In some embodiments, variants of the MAGE-A1 TCR or its antigen-binding domain with one or more amino acid substitutions are provided. Amino acid substitutions can be introduced into the relevant MAGE-A1 TCR or its antigen-binding domain and are used to screen products for desired activities, such as preserved / enhanced antigen binding or reduced immunogenicity.

[0167] Conservative substitutions are shown in Table 2 below.

[0168] Amino acids can be grouped into different categories based on their common side chain characteristics:

[0169] Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0170] Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0171] Acids: Asp, Glu;

[0172] Alkaline: His, Lys, Arg;

[0173] Residues that affect chain orientation: Gly, Pro;

[0174] Fang ethnic group: Trp, Tyr, Phe.

[0175] Non-conservative substitutions will necessarily involve replacing members of one of these categories with members of another. An illustrative example of a substitution variant is the affinity-matured antibody moiety, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. In short, it involves mutating one or more CDR residues to produce a variant antibody moiety on a phage and screening for specific biological activities (e.g., binding affinity).

[0176] Table 2: Conservative Substitution

[0177] Original residues Exemplary substitution Better alternative Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0178] In short, it involves mutating one or more CDR residues to produce a variant portion on the phage and screening for specific biological activities (e.g., binding affinity).

[0179] Alterations (e.g., substitutions) can be made in the MAGE-A1 TCR or its antigen-binding domain to, for example, increase affinity. Affinity maturation achieved through the construction of a secondary library and reselection from the secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-guided mutation induction). A secondary library is then generated. This library is then screened to identify any variants of the TCR or its antigen-binding domain with the desired affinity. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutation induction or modeling. Specifically, CDR3 is typically targeted.

[0180] In some embodiments, substitution, insertion, or deletion may occur within one or more CDRs, as long as these changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions as described herein) that do not substantially reduce binding affinity may be made in the TCR or its antigen-binding domain. In some embodiments of the variant TCR or its antigen-binding domain described above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0181] A suitable method for identifying antibody moieties that can be targeted by mutation-induced mutations is called "alanine-scan mutation induction," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the TCR or its antigen-binding domain and the MHC antigen complex is affected. Other substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, the crystal structure of the antigen-MHC-TCR moieties can be determined to identify contact points between the antibody moieties and the antigen. Such contact residues and adjacent residues can be targeted as substitution candidates or excluded from substitution candidate selection. Variants can be screened to determine if they contain the desired properties.

[0182] Amino acid sequence insertions include amino and / or carboxyl-terminal fusions ranging in length from one residue to one hundred or more residues of a polypeptide, as well as intra-sequence insertions having one or more amino acid residues. Examples of terminal insertions include TCR moieties having an N-terminal methionine residue. Other insertion variants of the TCR moieties include fusions of the N-terminus or C-terminus of the TCR moieties with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of an antibody moieties.

[0183] derivative

[0184] In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain, as described herein, may be further modified to contain additional non-protein portions known in the art and readily available. Suitable portions for the derivatization of the MAGE-A1 tumor-specific TCR or its antigen-binding domain 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-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and polydextrose or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / 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 can have any molecular weight and can be branched or unbranched. The number of polymers linked to the MAGE-A1 tumor-specific TCR or its antigen-binding domain can vary, and if more than one polymer is linked, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including (but not limited to) the specific properties or functions to be modified by the MAGE-A1 tumor-specific TCR or its antigen-binding domain, and whether the MAGE-A1 tumor-specific TCR or its antigen-binding domain derivative will be used for conditioned therapies.

[0185] In some embodiments, a conjugate is provided between the MAGE-A1 tumor-specific TCR or its antigen-binding domain and a non-protein portion that can be selectively heated by exposure to radiation. In some embodiments, the non-protein portion 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 portion to a temperature that kills cells proximal to the non-protein portion of the MAGE-A1 tumor-specific TCR.

[0186] V. Pharmaceutical Composition

[0187] This document also provides a composition (e.g., a pharmaceutical composition, also referred to herein as a formulation) comprising any of the MAGE-A1 tumor-specific TCRs or their antigen-binding domains according to the embodiments described herein, a nucleic acid encoding any of the MAGE-A1 tumor-specific TCRs or their antigen-binding domains according to the embodiments described herein, or MAGE-A1 tumor-specific TCR effector cells according to the embodiments described herein. In some embodiments, the composition is a MAGE-A1 tumor-specific TCR effector cell composition (e.g., a pharmaceutical composition) comprising effector cells (e.g., T cells) that present on a surface any of the MAGE-A1 tumor-specific TCRs or their antigen-binding domains according to the embodiments described herein. In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition is a pharmaceutical composition. The composition may comprise a homogeneous cell population or a heterogeneous cell population. The homogeneous cell population comprises MAGE-A1 tumor-specific TCR effector cells of the same cell type expressing the same MAGE-A1 tumor-specific TCR or its antigen-binding domain. The heterogeneous cell population comprises multiple populations of MAGE-A1 tumor-specific TCR effector cells of different cell types and / or expressing different MAGE-A1 tumor-specific TCRs or their antigen-binding domains. The composition may further comprise cells that are not MAGE-A1 tumor-specific TCR effector cells. Therefore, in some embodiments, a MAGE-A1 tumor-specific TCR effector cell composition is provided, comprising a homogeneous cell population of MAGE-A1 tumor-specific TCR effector cells (e.g., MAGE-A1 tumor-specific TCRT cells) of the same cell type expressing the same MAGE-A1 tumor-specific TCR. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition is a pharmaceutical composition.

[0188] In some embodiments, a MAGE-A1 tumor-specific TCR effector cell composition is provided, comprising a heterogeneous cell population containing multiple MAGE-A1 tumor-specific TCR effector cell populations, wherein the MAGE-A1 tumor-specific TCR effector cell populations contain different cell types and / or MAGE-A1 tumor-specific TCRs or their antigen-binding domains. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population has a cell type selected from cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, all MAGE-A1 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A1 tumor-specific TCR effector cells are cytotoxic T cells). In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population has a cell type different from other MAGE-A1 tumor-specific TCR effector cell populations (e.g., one MAGE-A1 tumor-specific TCR effector cell population consists of cytotoxic T cells and other MAGE-A1 tumor-specific TCR effector cell populations consist of natural killer T cells). In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses the same MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that is different from other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that is different from other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to the same target antigen. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen different from that of other MAGE-A1 tumor-specific TCR effector cell populations (e.g., one MAGE-A1 tumor-specific TCR effector cell population specifically binds to the pMHC complex and other MAGE-A1 tumor-specific TCR effector cell populations specifically bind to cell surface receptors).In some embodiments, at least one population of MAGE-A1 tumor-specific TCR effector cells expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to different target antigens, and each population of MAGE-A1 tumor-specific TCR effector cells expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen associated with the same disease or condition (e.g., each of the target antigens is associated with cancer, such as breast cancer). In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition is a pharmaceutical composition.

[0189] Therefore, in some embodiments, a MAGE-A1 tumor-specific TCR effector cell composition is provided, comprising a plurality of MAGE-A1 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein all MAGE-A1 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A1 tumor-specific TCR effector cells are cytotoxic T cells), and wherein each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that is different from other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to the same target antigen. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a target antigen MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a different target antigen than other MAGE-A1 tumor-specific TCR effector cell populations (e.g., one MAGE-A1 tumor-specific TCR effector cell population specifically binds to the pMHC complex and other MAGE-A1 tumor-specific TCR effector cell populations specifically bind to cell surface receptors). In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a different target antigen, and each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen associated with the same disease or condition (e.g., each of the target antigens is associated with cancer, such as breast cancer). In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition is a pharmaceutical composition.

[0190] In some embodiments, a composition is provided comprising a plurality of MAGE-A1 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein at least one MAGE-A1 tumor-specific TCR effector cell population has a cell type different from the other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, all MAGE-A1 tumor-specific TCR effector cell populations have completely different cell types. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population has a cell type selected from cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses the same MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain different from the other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that is different from other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to the same target antigen. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen that is different from other MAGE-A1 tumor-specific TCR effector cell populations (e.g., one MAGE-A1 tumor-specific TCR effector cell population specifically binds to the pMHC complex and other MAGE-A1 tumor-specific TCR effector cell populations specifically bind to cell surface receptors). In some embodiments, at least one population of MAGE-A1 tumor-specific TCR effector cells expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to different target antigens, and each population of MAGE-A1 tumor-specific TCR effector cells expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen associated with the same disease or condition (e.g., each of the target antigens is associated with cancer, such as breast cancer). In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition is a pharmaceutical composition.

[0191] At various points during composition preparation, cell cryopreservation may be necessary or appropriate. The terms "frozen / freezing" and "cryopreserved / cryopreserving" are used interchangeably. Freezing includes freeze-drying.

[0192] As those skilled in the art will understand, frozen cells can be destructive (see Mazur, P., 1977, Cryobiology 14:251-272), but numerous procedures exist to prevent such destruction. For example, destruction can be avoided by (a) using cryoprotectants, (b) controlling the freezing rate, and / or (c) storing at sufficiently low temperatures that minimize degradation responses. Examples of cryoprotectants include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), glycerol, polyvinylpyrrolidone (Rinfret, 1960, Ann. NYAcad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 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), and amino acids (Phan The Tran and Bender, 1960, Exp. Cell Res. 20:651), methanol, acetaminophen, glyceryl monoacetate (Lovelock, 1954, BioChem. J. 56:265), and inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender, 1961, Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, ed. Ilbery, Butterworth, London, p. 59). In certain embodiments, DMSO may be used. The protective effect of DMSO can be enhanced by adding plasma (e.g., to a concentration of 20-25%). After the addition of DMSO, cells can be held at 0°C until freezing, as a 1% concentration of DMSO can be toxic at temperatures above 4°C.

[0193] In cell cryopreservation, a slow, controlled cooling rate is crucial, and different cryoprotectants (Rapatz et al., 1968, Cryobiology 5(1):18-25) and different cell types have different optimal cooling rates (for the effect of cooling rate on stem cell survival and transplantation potential, see, for example, Rowe and Rinfret, 1962, Blood 20:636; Rowe, 5 1966, Cryobiology 3(1):12-18; Lewis et al., 1967, Transfusion 7(1):17-32; and Mazur, 1970, Science 168:939-949). The heat generated during the melting phase of water turning into ice should be minimal. Cooling programs can be implemented using, for example, programmable cryostats or methanol bath programs. Programmable cryostats allow for the determination of optimal cooling rates and facilitate standard reproducible cooling.

[0194] In a particular embodiment, DMSO-treated cells are pre-cooled on ice and transferred to a tray containing refrigerated methanol, which is then placed in a mechanical refrigeration unit (e.g., Harris or Revco) at -80°C. Thermocouple measurements of the methanol bath and the sample indicate a cooling rate of 1°C to 3°C / min, which is preferred. After at least two hours, the sample reaches a temperature of -80°C and can be placed directly in liquid nitrogen (-196°C). After thorough freezing, the cells can be rapidly transferred to long-term cryogenic storage containers.

[0195] In a preferred embodiment, the sample can be cryogenically stored in liquid nitrogen (-196°C) or vapor (-1°C). The availability of high-efficiency liquid nitrogen freezers facilitates such storage. Other considerations and procedures regarding handling, cryopreservation, and long-term cell storage can be found in the following illustrative references: U.S. Patents 4,199,022, 3,753,357, and 4,559,298; Gorin, 1986, Clinics In Haematology 15(1):19-48 Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 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.

[0196] After cryopreservation, the frozen cells can be thawed for use according to methods known to those skilled in the art. The frozen cells are preferably thawed rapidly and refrigerated immediately after thawing. In a particular embodiment, the vial containing the frozen cells can be immersed in a warm water bath up to its neck; gentle rotation ensures that the cell suspension mixes as it thaws and improves heat transfer from the warm water to the internal ice. Once the ice has completely melted, the vial can be placed on ice immediately.

[0197] In certain embodiments, methods to prevent cell aggregation may be used during thawing. Exemplary methods include adding deoxyribonuclease (Spitzer et al., 113, 1980, Cancer 45:3075-3085), low molecular weight polydextrose and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20:17-24), etc., before and / or after freezing. As those skilled in the art will understand, if a cryoprotectant that is toxic to humans is used, it should be removed before therapeutic use. DMSO is not seriously toxic.

[0198] Exemplary carriers and cell administration methods are described on pages 14-15 of U.S. Patent Publication No. 2010 / 0183564. Other pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by David B. Troy, Lippicott, Williams & Wilkins (2005).

[0199] In certain embodiments, cells may be collected from the culture medium, washed, and concentrated in a carrier to a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), Plasma-LyteA(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0200] In certain embodiments, the carrier may be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In certain embodiments, the infusion carrier comprises buffered saline containing 5% HAS or dextran. Other isotropic agents include polyhydroxy sugar alcohols, including triols or higher sugar alcohols such as glycerol, erythritol, arabinitol, xylitol, sorbitol, or mannitol.

[0201] The carrier may include buffers such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0202] Stabilizers are a broad class of excipients whose functions range from build-up agents to additives that help prevent cells from adhering to container walls. 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, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, and cyclic alcohols such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (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.

[0203] When needed or when advantageous, the composition may include a local anesthetic such as lidocaine to relieve pain at the injection site.

[0204] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethylbenzamide chloride, halogenated benzyl ammonium chloride, hexahydroxy quaternary ammonium chloride, alkylparaben esters (e.g., methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0205] The therapeutically effective amount of cells in 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 One cell or more than 10 11 Cells. In the compositions and formulations disclosed herein, the cell volume is generally one liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the applied cell density is generally greater than 10. 4 cells / mL, 10 7 Cells / mL or 10 8 Cells / mL

[0206] This document also provides MAGE-A1 tumor-specific TCR nucleic acid compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any of the nucleic acids encoding the MAGE-A1 tumor-specific TCR or its antigen-binding domain as described herein. In some embodiments, the MAGE-A1 tumor-specific TCR nucleic acid composition is a pharmaceutical composition. In some embodiments, the MAGE-A1 tumor-specific TCR nucleic acid composition further comprises any of the following: isotonic agents, excipients, diluents, thickeners, stabilizers, buffers, and / or preservatives; and / or aqueous mediators, such as purified water, glucose solutions, buffer solutions, physiological saline, polymer solutions, or ribonuclease-free water. The amounts of such additives and aqueous mediators to be added may be appropriately selected according to the intended use of the MAGE-A1 tumor-specific TCR nucleic acid composition.

[0207] The compositions and formulations disclosed herein can be prepared for administration by means of, for example, injection, infusion, perfusion, or irrigation. The compositions and formulations can be further formulated for intramedullary, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, local, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous injection.

[0208] Formulations intended for in vivo administration must be sterile. This requirement can be readily achieved, for example, by filtration via a sterile filter membrane. Treatment methods using the MAGE-A1 tumor-specific TCR or its antigen-binding domain can administer the MAGE-A1 tumor-specific TCR or its antigen-binding domain and / or composition of the present invention to an individual (e.g., a mammal, such as a human) to treat diseases and / or conditions associated with target antigen (TA) expression (also referred to herein as "target antigen positive" or "TA positive" diseases or conditions), including, for example, cancer. Therefore, in some embodiments, this application provides a method for treating an individual with a target antigen positive disease (e.g., cancer) comprising administering to the individual an effective amount of a composition (e.g., a pharmaceutical composition) comprising, for example, any of the MAGE-A1 tumor-specific TCR or its antigen-binding domain described herein.

[0209] In some embodiments, the composition further comprises cells (e.g., effector cells) associated with the MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, the cancer is selected from, for example, the following: adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, liver cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer, and thyroid cancer.

[0210] VI. Treatment methods

[0211] In some embodiments, a method of treating an individual with a target antigen-related disease (e.g., cancer) is provided, comprising administering to the individual an effective amount of a composition comprising a MAGE-A1 tumor-specific TCR or its antigen-binding domain according to any of the embodiments described herein, or a nucleic acid encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain according to any of the embodiments described herein, or effector cells (e.g., T cells or natural killer cells) presenting a MAGE-A1 tumor-specific TCR or its antigen-binding domain (e.g., a MAGE-A1 tumor-specific TCR or its antigen-binding domain) on a surface. In some embodiments, the composition comprises a) a first nucleic acid sequence encoding a first polypeptide chain, the 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, the 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, the 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, the 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, the 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, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40; and / or g) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 51 and h) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 54; and / or i) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 9. The amino acid sequence of SEQ ID NO:65 and j) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:68; and / or k) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:79 and l) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:82; and / or m) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:93 and n) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:96; and / or o) the first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:107 and p) the second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:110;And / or q) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:121 and r) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:124; and / or s) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:135 and t) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:138; and / or u) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:149 and v) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:152; and / or w) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:163 and x) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:166; and / or y) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:124. The amino acid sequence of SEQ ID NO:177 and z) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:180; and / or aa) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO:191 and ab) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO:194. In some embodiments, effector cells expressing MAGE-A1 tumor-specific TCR or its antigen-binding domain further comprise a P2A nucleic acid sequence encoding SEQ ID NOs:13, 27, 41, 55, 69, 83, 97, 111, 125, 139, 153, 167, 181, or 195. In some embodiments, an effector cell (e.g., a T cell) expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain on its surface is provided, comprising a nucleic acid sequence encoding one or more of the amino acid sequences selected from SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182, and 196.

[0212] In some embodiments, the above composition comprises an isolated MAGE-A1 TCR or its antigen-binding domain, including a) a TCRα chain comprising the amino acid sequence of SEQ ID NO:9 and b) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:12; and / or c) a TCRα chain comprising the amino acid sequence of SEQ ID NO:23 and d) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:26; and / or e) a TCRα chain comprising the amino acid sequence of SEQ ID NO:37 and f) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:40; and / or g) a TCRα chain comprising the amino acid sequence of SEQ ID NO:51 and h) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:54; and / or i) a TCRα chain comprising the amino acid sequence of SEQ ID NO:65 and j) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:68; and / or k) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:79 and l) a TCRβ chain comprising the amino acid sequence of SEQ ID NO:9. TCRβ chain containing the amino acid sequence of SEQ ID NO:82; and / or m) TCRα chain containing the amino acid sequence of SEQ ID NO:93 and n) TCRβ chain containing the amino acid sequence of SEQ ID NO:96; and / or o) TCRα chain containing the amino acid sequence of SEQ ID NO:107 and p) TCRβ chain containing the amino acid sequence of SEQ ID NO:110; and / or q) TCRα chain containing the amino acid sequence of SEQ ID NO:121 and r) TCRβ chain containing the amino acid sequence of SEQ ID NO:124; and / or s) TCRα chain containing the amino acid sequence of SEQ ID NO:135 and t) TCRβ chain containing the amino acid sequence of SEQ ID NO:138; and / or u) TCRα chain containing the amino acid sequence of SEQ ID NO:149 and v) TCRβ chain containing the amino acid sequence of SEQ ID NO:152; and / or w) TCRα chain containing the amino acid sequence of SEQ ID NO:163 and x) TCRβ chain containing the amino acid sequence of SEQ ID NO:82. The TCRβ chain containing the amino acid sequence of SEQ ID NO:166; and / or y) the TCRα chain containing the amino acid sequence of SEQ ID NO:177 and z) the TCRβ chain containing the amino acid sequence of SEQ ID NO:180; and / or aa) the TCRα chain containing the amino acid sequence of SEQ ID NO:191 and ab) the TCRβ chain containing the amino acid sequence of SEQ ID NO:194. In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain further comprises the P2A sequence of SEQ ID NOs:13, 27, 41, 55, 69, 83, 97, 111, 125, 139, 153, 167, 181 or 195.In some embodiments, a MAGE-A1 tumor-specific TCR or its antigen-binding domain is provided, comprising one or more amino acid sequences of SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196.

[0213] In some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in an individual of need is provided, comprising administering to the individual an effective amount of a composition comprising effector cells (e.g., T cells or natural killer cells) that present a MAGE-A1 tumor-specific TCR or its antigen-binding domain (e.g., a MAGE-A1 tumor-specific TCR or its antigen-binding domain) on a surface, wherein the target antigen of the effector cells (e.g., T cells) of the MAGE-A1 tumor-specific TCR is a MAGE-A1 peptide / MHC complex. In some embodiments, the MHC protein in the MAGE-A1 peptide / MHC complex is an MHC class II protein. In some embodiments, the MHC class II protein is HLA-DRA. In some embodiments, the MHC class II protein is HLA-DRB. In some embodiments, HLA-DRA is DRA*01:01. In some embodiments, HLA-DRB is DRB1*07:01. In some embodiments, HLA-DRB is DRB1*12:02. In some embodiments, the effector cells are γδ T cells. In some embodiments, the effector cells are αβ T cells. In some embodiments, the effector cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 257-287 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-287 Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283Peptide / MHCII DRA*01:01 / DRB1*12:02 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 269-283 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex. In some embodiments, the target antigen of effector cells (e.g., T cells) that express MAGE-A1 tumor-specific TCR or its antigen-binding domain on their surface is MAGE-A1. 265-279 Peptide / MHCII DRA*01:01 / DRB1*07:01 complex.

[0214] This invention also covers methods for treating target antigen-related diseases in individuals of need, comprising administering to the individual a composition comprising a plurality of effector cells expressing different MAGE-A1 tumor-specific TCRs or their antigen-binding domains. Thus, in some embodiments, according to any of the methods described herein for treating target antigen-related diseases in individuals, the composition is a heterogeneous MAGE-A1 tumor-specific TCR effector cell composition as described herein. For example, in some embodiments, a method of treating an individual with a target antigen-related disease (e.g., cancer) is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A1 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A1 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein all MAGE-A1 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A1 tumor-specific TCR effector cells are cytotoxic T cells), wherein each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR different from other MAGE-A1 tumor-specific TCR effector cell populations, and wherein at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR specifically binding to a target antigen. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a different target antigen. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a different target antigen, each of which is associated with a target antigen-related disease.

[0215] In some embodiments, a method of treating an individual with a target antigen-related disease (e.g., cancer) is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A1 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A1 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein at least one MAGE-A1 tumor-specific TCR effector cell population has a cell type different from other MAGE-A1 tumor-specific TCR effector cell populations, and wherein at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen. In some embodiments, all MAGE-A1 tumor-specific TCR effector cell populations are of different cell types. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population has a cell type selected from cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses the same MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that is different from other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that is different from other MAGE-A1 tumor-specific TCR effector cell populations. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a target antigen. In some embodiments, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to a different target antigen. In some implementations, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to different target antigens, each of which is associated with a target antigen-related disease.

[0216] In some embodiments, a method of treating an individual in need of a disease associated with multiple target antigens is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A1 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A1 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein all MAGE-A1 tumor-specific TCR effector cells in the composition have the same cell type (e.g., all MAGE-A1 tumor-specific TCR effector cells are cytotoxic T cells), wherein each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that differs from other MAGE-A1 tumor-specific TCR effector cell populations, and wherein, for each of the plurality of target antigens, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that specifically binds to the target antigen. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some implementations, the MAGE-A1 tumor-specific TCR effector cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

[0217] In some embodiments, a method of treating an individual in need of a disease associated with multiple target antigens is provided, comprising administering to the individual an effective amount of a heterogeneous MAGE-A1 tumor-specific TCR effector cell composition comprising a plurality of MAGE-A1 tumor-specific TCR effector cell populations according to any of the embodiments described herein, wherein at least one MAGE-A1 tumor-specific TCR effector cell population has a cell type different from other MAGE-A1 tumor-specific TCR effector cell populations, and wherein, for each of the plurality of target antigens, at least one MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain specifically binding to the target antigen. In some embodiments, all MAGE-A1 tumor-specific TCR effector cell populations are of different cell types. In some embodiments, the MAGE-A1 tumor-specific TCR effector cells are T cells. In some embodiments, each MAGE-A1 tumor-specific TCR effector cell population has a cell type selected from cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some implementations, each MAGE-A1 tumor-specific TCR effector cell population expresses a MAGE-A1 tumor-specific TCR or its antigen-binding domain that differs from other MAGE-A1 tumor-specific TCR effector cell populations.

[0218] 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 individual is a human. In some embodiments, the individual is a clinical patient, clinical trial volunteer, laboratory animal, etc. In some embodiments, the individual is less than about 60 years of age (including, for example, less than about 50, 40, 30, 25, 20, 15, or 10 years of age). In some embodiments, the individual is more than about 60 years of age (including, for example, more than about 70, 80, 90, or 100 years of age). In some embodiments, the individual is diagnosed with or is environmentally or genetically predisposed to one or more of the diseases or conditions 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.

[0219] In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition of the present invention is administered in combination with a second, third, or fourth agent (including, for example, an anti-hypertrophic agent, a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent) to treat diseases or conditions involving target antigen expression. In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition is administered in combination with a cytokine (e.g., IL-2). In some embodiments, the MAGE-A1 tumor-specific TCR or its antigen-binding domain is administered in combination with an agent that increases MHC protein expression and / or enhances MHC protein surface presentation to peptides. In some embodiments, the agent includes, for example, an IFN receptor agonist, an Hsp90 inhibitor, a p53 expression enhancer, and a chemotherapeutic agent. In some embodiments, the agent is an IFN receptor agonist, including, for example, IFNγ, IFNβ, and IFNα. In some embodiments, the agent is an Hsp90 inhibitor, including, for example, tanespimycin (17-AAG), alvespimycin (17-DMAG), retasimicin (IPI-504), IPI-493, CNF2024 / BIIB021, MPC-3100, Debio0932 (CUDC-305), PU-H71, 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, etoposide, cisplatin, paclitaxel, and vinblastine. In some embodiments, a method of treating a target antigen-positive disease in an individual of need is provided, comprising administering to the individual a MAGE-A1 tumor-specific TCR effector cell composition according to any of the embodiments described herein, along with a cytokine (e.g., IL-2). In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition and the cytokine are administered simultaneously. In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition and the cytokine are administered sequentially.

[0220] In some embodiments, a method is provided for treating a target antigen-positive disease in an individual of need, wherein cells expressing the target antigen typically do not present or present at relatively low levels on their surface a complex comprising the target antigen and MHCI-like proteins. The method comprises administering to the individual a MAGE-A1 tumor-specific TCR effector cell composition according to any of the embodiments described herein, and agents that enhance the expression of MHCI-like proteins and / or enhance the surface presentation of MHCI-like proteins to the target antigen. In some embodiments, the agents include, for example, IFN receptor agonists, Hsp90 inhibitors, p53 expression enhancers, and chemotherapeutic agents. In some embodiments, the agents are IFN receptor agonists, including, for example, IFNγ, IFNβ, and IFNα. In some embodiments, the agent is an Hsp90 inhibitor, including, for example, spiramycin (17-AAG), aspiramycin (17-DMAG), retamycin (IPI-504), IPI-493, CNF2024 / BIIB021, MPC-3100, Debio0932 (CUDC-305), PU-H71, galitepi (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 vincristine. In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition and the drug are administered simultaneously. In some embodiments, the MAGE-A1 tumor-specific TCR effector cell composition and the drug are administered sequentially.

[0221] In some embodiments, a method of treating a target antigen-related disease (e.g., cancer) in an individual of need is provided, comprising administering to the individual an effective amount of a composition comprising a nucleic acid encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain according to any of the embodiments described herein. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0222] Cancer treatment can be assessed by, for example, tumor regression, tumor weight or size reduction, percentage of 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 the efficacy of a therapy can be used, including, for example, measuring the response via radiographic imaging.

[0223] In some embodiments, therapeutic efficacy is measured as a percentage of tumor growth inhibition (TGI%), calculated using the formula 100 - (T / C × 100), where T is the mean relative tumor volume of the treated tumor and C is the mean 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%.

[0224] In some embodiments, therapeutic efficacy is measured as a percentage of tumor growth inhibition (TGI%), calculated using the formula 100 - (T / C × 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%.

[0225] In some implementations, any of the methods for treating an individual’s target antigen-related disease as described herein are further combined with immunosuppressants, therapeutic antibodies, chemotherapy, radiation therapy, surgery, or any combination thereof.

[0226] disease

[0227] In some embodiments, MAGE-A1 tumor-specific TCR effector cells can be used to treat cancers associated with a target antigen. Cancers that can be treated using any of the methods described herein include non-vascularized tumors, or tumors that are not yet substantially vascularized, as well as vascularized tumors. Cancers may comprise non-solid tumors (e.g., hematologic malignancies, such as leukemia and lymphoma) or may comprise solid tumors. Types of cancers to be treated with the MAGE-A1 tumor-specific TCR effector cells of the present invention include (but are not limited to) carcinomas, blastomas, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included. Hematologic malignancies are cancers of the blood or bone marrow. Examples of blood (or hematologic) cancers include leukemia, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (refractory and advanced forms), multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodyplasia syndrome, hairy cell leukemia, and myelodyplasia. Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma).Examples of solid tumors (such as sarcomas and carcinomas) include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, gastric cancer, malignant lymphoma, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid cancer (such as medullary thyroid carcinoma and papillary thyroid carcinoma), pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, and branchiocarcinoma. Tracheal cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, Wilms' tumor, cervical cancer (e.g., cervical cancer and pre-invasive cervical dysplasia), colorectal cancer, anal cancer, anal canal cancer or anorectal cancer, vaginal cancer, vulvar cancer (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma), penile cancer, oropharyngeal cancer, esophageal cancer, head cancer (e.g., squamous cell carcinoma), cervical cancer (e.g., squamous cell carcinoma), testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, Leydig cell tumor). Tumors, fibromas, fibroadenomas, adenomatous tumors and lipomas, bladder cancer, kidney cancer, melanoma, uterine cancer (e.g., endometrial cancer), urethral epithelial cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral cancer and bladder cancer), and CNS tumors (e.g. gliomas (e.g., brainstem glioma and mixed glioma), glioblastoma (also known as polymorphic glioblastoma), astrocytoma, CNS lymphoma, germ cell tumor, neuroblastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

[0228] Cancer treatment can be assessed, for example, by tumor regression, tumor weight or size reduction, 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 the efficacy of a therapy can be used, including, for example, measuring the response via radiographic imaging.

[0229] Products and reagent kits

[0230] In some embodiments of the invention, an article is provided containing materials suitable for treating target antigen-positive diseases such as cancers (e.g., adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, 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). The article may include a container and markings or instructions for use on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from various materials, such as glass or plastic. Generally, the container contains a composition that can effectively treat the diseases or conditions described herein and may have sterile inlet / outlet ports (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one active agent in the composition is an effector cell that presents the MAGE-A1 tumor-specific TCR or its antigen-binding domain of the present invention on a surface. Labelling or product information indicates that the composition is intended for the treatment of a specific condition. Labelling or product information will further include instructions for administering the MAGE-A1 tumor-specific TCR effector cell composition to a patient. Articles and kits comprising the combination therapies described herein are also covered.

[0231] A drug package insert refers to the information typically included in the packaging of commercially available therapeutic products, containing information about indications, uses, dosage, administration, contraindications, and / or warnings related to the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is used to treat target antigen-positive cancers (e.g., adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, 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 product may further include a second container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other substances required from a commercial and user perspective, including other buffer solutions, diluents, filters, needles, and syringes.

[0232] Kits are also provided for various purposes, such as for treating target antigen-positive diseases or conditions described herein, in combination with products as appropriate. Kits of the present invention comprise one or more containers containing a MAGE-A1 tumor-specific TCR effector cell composition (or unit dosage form and / or product), and in some embodiments, further comprising another agent (as described herein) according to any of the methods described herein and / or instructions for use. Kits may further include instructions for selecting individuals suitable for treatment. The instructions provided in the kits of the present invention are written instructions typically found on markings or pharmaceutical instructions (e.g., paper sheets included in the kit), but machine-readable instructions (e.g., instructions contained on a magnetized or optical storage disk) are also acceptable.

[0233] For example, in some embodiments, the kit comprises a composition comprising effector cells that surface-present MAGE-A1 tumor-specific TCRs. In some embodiments, the kit comprises a) a composition comprising effector cells that surface-present MAGE-A1 tumor-specific TCRs or their antigen-binding domains, and b) an effective amount of at least one other agent, wherein the other agent increases the expression of MHC proteins and / or enhances the surface presentation of MHC proteins (e.g., IFNγ, IFNβ, IFNα, or Hsp90 inhibitors) to peptides. In some embodiments, the kit comprises a) a composition comprising effector cells that surface-present MAGE-A1 tumor-specific TCRs or their antigen-binding domains, and b) administration to an individual.

[0234] In some embodiments, the kit comprises a) a composition containing effector cells that surface-present MAGE-A1 tumor-specific TCR or its antigen-binding domain, b) an effective amount of at least one other agent, wherein the other agent increases the expression of MHC proteins and / or enhances the surface presentation of MHC proteins (e.g., IFNγ, IFNβ, IFNα, or Hsp90 inhibitors) to peptides, and c) instructions for administering the MAGE-A1 tumor-specific TCR effector cell composition and other agents to an individual for the treatment of target cell-positive diseases (e.g., cancer). The MAGE-A1 tumor-specific TCR effector cell composition and other agents may be present in separate containers or a single container. For example, the kit may contain a single composition or two or more compositions, wherein one composition contains MAGE-A1 tumor-specific TCR effector cells and another composition contains another agent.

[0235] In some embodiments, the kit comprises a) a composition containing a MAGE-A1 tumor-specific TCR or its antigen-binding domain, and b) a specification relating to the combination of a MAGE-A1 tumor-specific TCR or its antigen-binding domain with effector cells (e.g., effector cells derived from an individual, such as T cells or natural killer cells) to form an effector cell comprising an MAGE-A1 tumor-specific TCR or its antigen-binding domain presented on a surface, and the administration of the MAGE-A1 tumor-specific TCR effector cell composition to an individual for the treatment of a target antigen-positive disease (e.g., cancer). In some embodiments, the kit comprises a) a composition containing a MAGE-A1 tumor-specific TCR or its antigen-binding domain, and b) effector cells (e.g., cytotoxic cells). In some embodiments, the kit comprises a) a composition containing a MAGE-A1 tumor-specific TCR or its antigen-binding domain, b) effector cells (e.g., cytotoxic cells), and c) a specification relating to the combination of a MAGE-A1 tumor-specific TCR or its antigen-binding domain with effector cells to form an effector cell comprising presenting a MAGE-A1 tumor-specific TCR or its antigen-binding domain on a surface, and the administration of the MAGE-A1 tumor-specific TCR effector cell composition to an individual for the treatment of a target antigen-positive disease (e.g., cancer).

[0236] In some embodiments, the kit comprises a nucleic acid (or a set of nucleic acids) encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain. In some embodiments, the kit comprises a) a nucleic acid (or a set of nucleic acids) encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain, and b) a host cell (e.g., effector cells) for expressing the nucleic acid (or the set of nucleic acids). In some embodiments, the kit comprises a) a nucleic acid (or a set of nucleic acids) encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain, and b) a specification relating to i) expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain in host cells (e.g., effector cells, such as T cells), ii) preparing a composition comprising host cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain, and iii) administering to an individual the composition comprising host cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain to treat a target antigen-positive disease (e.g., cancer). In some embodiments, the host cells are derived from an individual. In some embodiments, the kit comprises a) a nucleic acid (or a set of nucleic acids) encoding a MAGE-A1 tumor-specific TCR or its antigen-binding domain, b) host cells (e.g., effector cells) for expressing the nucleic acid (or the set of nucleic acids), and c) instructions relating to i) expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain in host cells, ii) preparing a composition comprising host cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain, and iii) administering to an individual the composition comprising host cells expressing a MAGE-A1 tumor-specific TCR or its antigen-binding domain to treat a target antigen-positive disease (e.g., cancer). The kit of the present invention is in suitable packaging. Suitable packaging includes (but is not limited to) vials, bottles, jars, plastic packaging (e.g., sealed Mylar or plastic bags) and the like. Additional components, such as buffers, and illustrative information may be provided as appropriate with the kit. Articles of manufacture, including vials (e.g., sealed vials), bottles, jars, plastic packaging, etc., are also provided in this application.

[0237] Instructions for use related to the MAGE-A1 tumor-specific TCR effector cell composition generally include information about the desired therapeutic dose, duration of administration, and route of administration. Containers may be unit dose, bulk (e.g., multi-dose packs), or subunit dose. For example, kits containing sufficient doses of the MAGE-A1 tumor-specific TCR effector cell composition as disclosed herein may be provided to provide individuals with effective treatment for extended periods, such as one week, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, three weeks, four weeks, six weeks, eight weeks, three months, four months, five months, seven months, eight months, nine months, or longer.

[0238] The kit may also include multiple unit doses of MAGE-A1 tumor-specific TCR or its antigen-binding domain, a pharmaceutical composition, and instructions for use, packaged in sufficient quantities for storage and use in pharmacies, such as hospital pharmacies and dispensing facilities.

[0239] Those skilled in the art will recognize that various embodiments are possible within the scope and spirit of this invention. The invention will now be described in more detail with reference to the following non-limiting embodiments. The following embodiments further illustrate the invention, but should not be construed as limiting its scope in any way. It should be understood that one, some, or all of the various embodiments described herein can be combined to form other embodiments of the invention. Example

[0240] The following embodiments are intended purely as examples of this patent application and should not be construed as limiting the invention in any way. The following embodiments and detailed descriptions are provided by way of illustration and not by way of limitation.

[0241] Example 1: Preparation of MAGE-A1 specific T cells

[0242] Five MAGE-A1 antigen-specific T-cell receptors, ST01C05, 35C11, 35D09, ST01G05, and ST01F06, were prepared for characterization and validation experiments. The HLA and epitopes of these five tumor antigen-specific T-cell receptors are as follows: Figure 1A As shown. For each tumor antigen-specific T cell receptor, corresponding human (i.e., wild-type) TCR constructs and murine TCR constructs were prepared; codon optimization was performed for ST01C05, 35C11, 35D09, and ST01G05. The murine TCR constructs possess murine constant domains (mCα and mCβ1). Schematic diagrams of the wild-type, murine, and murine codon-optimized constructs of tumor antigen-specific T cell receptors are shown below. Figure 1B As shown.

[0243] Example 2: Validation of MAGE-A1-specific T cell receptor ST01C05

[0244] The purpose of this study is to verify the expression, peptide recognition, epitope recognition, HLA blockade, and in vitro toxicity of T-cell receptor (TCR) ST01C05.

[0245] TCR Expression and Peptide Recognition Assay

[0246] Methods for measuring TCR expression and peptide recognition, such as Figure 2A As shown.

[0247] TCR expression was assessed using a FACS assay to detect the Vβ chain of stained TCR cells. Briefly, TCR-transferred T cells were collected, washed with 10 mL of PBS (containing 2% fetal bovine serum), centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell clumps were resuspended by adding PBS (containing 2% fetal bovine serum) and adjusted to approximately 2 × 10⁶ cells / mL. 6 Cells / mL. Collect 100 μL / sample cell suspension and use it for TCR Vβ chain surface staining and detection by FACS.

[0248] The recognition of MAGE-A1 by TCR-transduced T cells was determined using the MAGE-A1257-287 peptide assay. TCR-transduced T cells were collected, washed with 10 mL of PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The TCR-transduced T cells were then resuspended in AIM-V medium (containing 10% fetal bovine serum) and adjusted to a concentration of 2 × 10⁶ cells / mL. 6 1 cell / mL or 5×10 5 Cells / mL were added to 96-well plates. MAGE-A1257-287 peptide was mixed with tumor-specific T cells, and 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-γ and CD3 staining, which was detected by FACS. For IFN-γ ELISA detection, 100 μL / well of LCL (1×10⁻⁶) was added. 5 100 μL / well of TCR-transduced T cells (5 × 10⁶ cells / mL) and 100 μL / well of TCR-transduced T cells (5 × 10⁶ cells / mL). 5 (1 cell / mL) was mixed into a 96-well plate and incubated for 24 hours. After 24 hours, 175 μL of supernatant was collected from each well of the 96-well plate and used for IFN-γ ELISA detection using the IFN-γ ELISA HRP kit.

[0249] like Figure 2B-2C As shown, ST01C05 can specifically recognize the 30-amino acid peptide MAGE-A1.257-287 .

[0250] LCL stimulation assay is used to verify antigen epitope recognition.

[0251] LCL stimulation assays were performed as follows. First, LCL was loaded with peptides: LCL was collected in 15 mL tubes, washed with 10 mL PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell clumps were resuspended in culture medium (RPMI 1640 containing 10% FBS) and adjusted to 1×10⁻⁶. 6 Cells / mL. Tumor antigen peptides were added to LCL to a final concentration of 5 μg / mL and cultured for 8 to 24 hours. TCR-T cells were then stimulated with peptide-loaded LCL as follows: The peptide-loaded LCL was collected and washed with 10 mL of PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The LCL was resuspended by adding AIM-V medium (containing 10% fetal bovine serum) and adjusted to 1 × 10⁶ cells / mL. 6 Cells / mL or 1×10 5 Cells / mL. TCR-transduced T cells were collected and washed with 10 mL of PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The TCR-transduced T cells were then resuspended in AIM-V medium (containing 10% fetal bovine serum) and adjusted to 2 × 10⁶ cells / mL or 5 × 10⁵ cells / mL. 100 μL / well of LCL (1 × 10⁶ cells / mL) was added. 6 100 μL / well of TCR-transduced T cells (2 × 10⁶ cells / mL) and 100 μL / well of TCR-transduced T cells (2 × 10⁶ cells / mL). 6 Cells were mixed in a 96-well dish with 100 μL / well of LCL (1 × 10⁻⁶ cells / mL). Brefeldin A (final concentration 3 μg / mL) was added to the wells and the cells were incubated for 4 hours. After 4 hours, cells were harvested and used for intracellular IFN-γ and CD3 staining, which was then detected by FACS. For IFN-γ ELISA detection, 100 μL / well of LCL (1 × 10⁻⁶ cells / mL) was added. 5 100 μL / well of TCR-transduced T cells (5 × 10⁶ cells / mL) and 100 μL / well of TCR-transduced T cells (5 × 10⁶ cells / mL). 5 (1 cell / mL) was mixed into a 96-well plate and incubated for 24 hours. After 24 hours, 175 μL of supernatant was collected from each well of the 96-well plate and used for IFN-γ ELISA detection using the IFN-γ ELISA HRP kit.

[0252] The peptide sequences of different epitopes of MAGE-A1 used in the experiments are as follows: Figure 3 As shown. LCL stimulation assays showed that, except for MAGE-A1 257-287In addition, ST01C05 can also identify the table position ETSYVKVLEYVIKVS(MAGE-A1) 273-287 (SEQ ID NO:366).

[0253] HLA restriction assay

[0254] Infuse K562 cells with various HLA-II genotypes with MAGE-A1 257-287 The peptide was used to stimulate T cells transfected with the T cell receptor in an HLA restriction assay. The remaining steps were the same as for the LCL stimulation assay, and the final measurement was performed using a human IFN-γ ELISA.

[0255] On Day 1, the high protein binding ELISA discs were plated with antibody 1-D1K (IFN-γ ELISA HRP kit) diluted to 2 μg / mL in PBS (pH 7.4) (by adding 50 μL / well) and incubated overnight at 4°C. On Day 2, the discs were washed twice with PBS (200 μL / well). The discs were then blocked by adding 200 μL / well of culture medium and incubated at room temperature (RT) for 1 hour. Human IFN-γ standards (IFN-γ ELISA HRP kit) were prepared in 2 mL PBS (with 1% BSA) to a concentration of 0.5 μg / mL and placed at RT for 15 minutes, followed by vortexing. 50 μL / well of sample or standard was diluted in culture medium and incubated at RT for 2 hours. Samples and standard probes were tested in duplicate. The discs were washed five times with PBS containing 0.05% Tween 20. Add 50 μL / well of antibody 7-B6-1-biotin (IFN-γ ELISA HRP kit) at 1 μg / mL (in PBS), incubate at RT for 1 hour, and wash. Add 50 μL / well of streptavidin-HRP (IFN-γ ELISA HRP kit) diluted 1:1000 in PBS, incubate at RT for 1 hour, and wash. Add 100 μL / well of TMB acceptor solution, incubate at RT in the dark for 15 to 30 minutes, until the solution in the wells turns visible blue. Add 50 μL / well of stop solution to stop the enzyme reaction. The solution color changes from blue to yellow. Measure the light intensity at 450 nm using an ELISA reader.

[0256] like Figure 4 As shown, T cells transfected with ST01C05 can recognize MAGE-A1 presented by DRA*01:01 / DRB1*12:02. 257-287 Antigenic peptides.

[0257] In vitro toxicity assay

[0258] T cells transfected with the T cell receptor ST01C05 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were transfected with DRA*01:01 / DRB1*12:02 and loaded with MAGE-A1. 257-287 293 T cells containing the antigen peptide. Untransfected T cell receptor T cells were mixed with target cells in the same proportion as a control group. The killing power of effector cells against target cells in both the experimental and control groups was continuously measured over 48 hours.

[0259] like Figure 5 As shown, T cells transfected with ST01C05 showed resistance to MAGE-A1-loaded cells. 257-287 The antigenic peptide exhibited strong cytotoxic activity against 293T target cells, killing 94.14% of the target cells. In the control group, untransfected T cells showed no cytotoxic effect on target cells.

[0260] Next, T cells transfected with the T cell receptor ST01C05 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were 293T cells transfected with DRA*01:01 / DRB1*12:02 and loaded with MAGE-A1 protein. T cells not transfected with the T cell receptor were mixed with target cells at the same ratio as the control group. The killing effector cells of the experimental and control groups on target cells were continuously measured over 48 hours.

[0261] like Figure 6 As shown, T cells transfected with ST01C05 exhibited a strong killing effect on 293T target cells carrying MAGE-A1 protein, with 98.48% of the target cells being killed. In the control group, untransfected T cells showed no killing effect on the target cells.

[0262] In another experiment, T cells transfected with the T cell receptor ST01C05 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were A549 cells transfected with DRA*01:01 / DRB1*12:02 and loaded with MAGE-A1 protein. T cells not transfected with the T cell receptor were mixed with target cells at the same ratio as the control group. The killing effector cells of the experimental and control groups on target cells were continuously measured over 48 hours.

[0263] like Figure 7 As shown, T cells transfected with ST01C05 significantly killed 293T target cells carrying MAGE-A1 protein, with 52.29% of the target cells being killed. In the control group, untransfected T cells had no killing effect on the target cells.

[0264] Example 3: Validation of MAGE-A1-specific T cell receptor 35C11

[0265] The purpose of this study is to validate the expression, peptide recognition, epitope recognition, HLA blockade, and in vitro toxicity of T-cell receptor (TCR) 35C11.

[0266] TCR Expression and Peptide Recognition Assay

[0267] Methods for measuring TCR expression and peptide recognition, such as Figure 8A As shown. The specific experimental method is the same as in Example 2.

[0268] like Figures 8B-8C As shown, ST01C05 can specifically recognize the 30-amino acid peptide MAGE-A1. 257-287 .

[0269] LCL stimulation assay is used to verify antigen epitope recognition.

[0270] The peptide sequences of different epitopes of MAGE-A1 used in the experiments are as follows: Figure 9 As shown. The specific experimental method is the same as in Example 2. LCL stimulation assays showed that, except for MAGE-A1 257-287 In addition, 35C11 can also identify the epitope RALAETSYVKVLEYV(MAGE-A1) 269-283 (SEQ ID NO:367).

[0271] HLA restriction assay

[0272] The specific experimental method is the same as in Example 2. Figure 10 As shown, T cells transfected with 35C11 can recognize MAGE-A1 presented by DRA*01:01 / DRB1*07:01. 257-287 Antigenic peptides.

[0273] In vitro toxicity assay

[0274] T cells transfected with T cell receptor 35C11 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were transfected with DRA*01:01 / DRB1*07:01 and loaded with MAGE-A1. 257-287 293 T cells containing the antigen peptide. Untransfected T cell receptor T cells were mixed with target cells in the same proportion as a control group. The killing power of effector cells against target cells in both the experimental and control groups was continuously measured over 48 hours.

[0275] like Figure 11 As shown, T cells transfected with 35C11 showed resistance to MAGE-A1-loaded cells. 257-287 The antigenic peptide exhibited strong cytotoxic activity against 293T target cells, killing 80.63% of the target cells. In the control group, untransfected T cells showed no cytotoxic effect on target cells.

[0276] Example 4: Validation of MAGE-A1-specific T cell receptor 35D09

[0277] The purpose of this study is to verify the expression, peptide recognition, epitope recognition, HLA blockade, and in vitro toxicity of T-cell receptor (TCR) 35D09.

[0278] TCR Expression and Peptide Recognition Assay

[0279] Methods for measuring TCR expression and peptide recognition, such as Figure 12A As shown. The specific experimental method is the same as in Example 2.

[0280] like Figure 12B-12C As shown, ST01C05 can specifically recognize the 30-amino acid peptide MAGE-A1. 257-287 .

[0281] LCL stimulation assay is used to verify antigen epitope recognition.

[0282] The peptide sequences of different epitopes of MAGE-A1 used in the experiments are as follows: Figure 13 As shown. The specific experimental method is the same as in Example 2. LCL stimulation assays showed that, except for MAGE-A1 257-287 In addition, 35D09 can also identify the epitope RALAETSYVKVLEYVIKVS(MAGE-A1) 269-287 (SEQ ID NO:368). Simultaneously, 35D09 corresponds to the epitope RALAETSYVKVLEYV(MAGE-A1). 269-283 (SEQ ID NO:367) has a certain recognition capability.

[0283] HLA restriction assay

[0284] The specific experimental method is the same as in Example 2. Figure 14 As shown, T cells transfected with 35D09 can recognize MAGE-A1 presented by DRA*01:01 / DRB1*12:02. 257-287 Antigenic peptides.

[0285] In vitro toxicity assay

[0286] T cells transfected with T cell receptor 35D09 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were transfected with DRA*01:01 / DRB1*12:02 and loaded with MAGE-A1. 257-287 293 T cells containing the antigen peptide. Untransfected T cell receptor T cells were mixed with target cells in the same proportion as a control group. The killing power of effector cells against target cells in both the experimental and control groups was continuously measured over 48 hours.

[0287] like Figure 15 As shown, T cells transfected with 35D09 showed resistance to MAGE-A1-loaded cells. 257-287 The antigenic peptide exhibited strong cytotoxic activity against 293T target cells, killing 99.94% of the target cells. In the control group, untransfected T cells showed no cytotoxic effect on target cells.

[0288] Example 5: Validation of MAGE-A1-specific T cell receptor ST01G05

[0289] The purpose of this study is to verify the expression, peptide recognition, epitope recognition, HLA blockade, and in vitro toxicity of T-cell receptor (TCR) ST01G05.

[0290] TCR Expression and Peptide Recognition Assay

[0291] Methods for measuring TCR expression and peptide recognition, such as Figure 16A As shown. The specific experimental method is the same as in Example 2.

[0292] like Figure 16B-16C As shown, ST01C05 can specifically recognize the 30-amino acid peptide MAGE-A1. 257-287 .

[0293] LCL stimulation assay is used to verify antigen epitope recognition.

[0294] The peptide sequences of different epitopes of MAGE-A1 used in the experiments are as follows: Figure 17 As shown. The specific experimental method is the same as in Example 2. LCL stimulation assays showed that, except for MAGE-A1 257-287 In addition, ST01G05 can also identify the table position LWGPRALAETSYVKV(MAGE-A1) 265-279 (SEQ ID NO:369).

[0295] HLA restriction assay

[0296] The specific experimental method is the same as in Example 2. Figure 18 As shown, T cells transfected with ST01G05 can recognize MAGE-A1 presented by DRA*01:01 / DRB1*07:01. 257-287 Antigenic peptides.

[0297] In vitro toxicity assay

[0298] T cells transfected with the T cell receptor ST01G05 were used as effector cells and mixed with target cells at a ratio of 5:1 as the experimental group. The target cells used in this experiment were transfected with DRA*01:01 / DRB1*07:01 and loaded with MAGE-A1. 257-287293 T cells containing the antigen peptide. Untransfected T cell receptor T cells were mixed with target cells in the same proportion as a control group. The killing power of effector cells against target cells in both the experimental and control groups was continuously measured over 48 hours.

[0299] like Figure 19 As shown, T cells transfected with ST01G05 showed resistance to MAGE-A1-loaded cells. 257-287 The antigenic peptide exhibited strong cytotoxic activity against 293T target cells, killing 89.74% of the target cells. In the control group, untransfected T cells showed no cytotoxic effect on target cells.

[0300] Example 6: Validation of MAGE-A1-specific T cell receptor ST01F06

[0301] The purpose of this study is to verify the expression, peptide recognition, epitope recognition, HLA blockade, and in vitro toxicity of T-cell receptor (TCR) ST01F06.

[0302] TCR Expression and Peptide Recognition Assay

[0303] Methods for measuring TCR expression and peptide recognition, such as Figure 20A As shown. The specific experimental method is the same as in Example 2.

[0304] like Figure 20B-20C As shown, ST01F06 can specifically recognize the 30-amino acid peptide MAGE-A1. 257-287 .

[0305] LCL stimulation assay is used to verify antigen epitope recognition.

[0306] The peptide sequences of different epitopes of MAGE-A1 used in the experiments are as follows: Figure 21 As shown. The specific experimental method is the same as in Example 2. LCL stimulation assays showed that, except for MAGE-A1 257-287 In addition, ST01F06 can also identify the epitope RALAETSYVKVLEYV(MAGE-A1) 269-283 (SEQ ID NO:367).

[0307] HLA blocking assay

[0308] HLA blocking assays were performed as follows. Peptide-loaded LCL was collected after LCL stimulation, washed with 10 mL PBS, centrifuged at 350 g for 5 minutes, and the supernatant was completely aspirated. The cell clumps were resuspended by adding AIM-V medium (containing 10% fetal bovine serum) and adjusted to a concentration of 1 × 10⁶ cells / mL. 5Cells / mL. Add 100 μL / well of cell suspension to a 96-well plate and add HLA blocking antibody (final concentration 50 μg / mL) to the appropriate wells, and incubate for 2 hours. Collect TCR-transferred T cells, wash with 10 mL of PBS, centrifuge at 350 g for 5 minutes, and completely aspirate the supernatant. Collect cell clumps by adding AIM-V medium (containing 10% fetal bovine serum) and adjust to 5 × 10⁶ cells / mL. 5 Cells / mL. Add 100 μL / well of TCR-transduced cell suspension to the corresponding well, mix with LCL, and incubate for 24 hours. After 24 hours, collect 175 μL of supernatant from each well in a 96-pan tray and subject it to IFN-γ ELISA using the IFN-γ ELISA HRP kit.

[0309] like Figure 22 As shown, the function of T cells transfected with ST01F06 is limited to HLA-DR.

[0310] HLA restriction assay

[0311] The specific experimental method is the same as in Example 2. Figure 23 As shown, T cells transfected with ST01T06 can recognize MAGE-A1 presented by DRA*01:01 / DRB1*07:01. 257-287 Antigenic peptides.

[0312] CDR sequence of an exemplary TCR

[0313]

[0314]

[0315] sequence list

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

Claims

1. An isolated tumor-specific T-cell receptor (TCR) that specifically binds to the major tissue compatibility complex (MHC) / MAGE-A1 epitope complex or its antigen-binding domain, said tumor-specific TCR comprising: The TCRα chain comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 85, CDR2 of the amino acid sequence shown in SEQ ID NO: 86, and CDR3 of the amino acid sequence shown in SEQ ID NO: 87; and the TCRβ chain comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 88, CDR2 of the amino acid sequence shown in SEQ ID NO: 89, and CDR3 of the amino acid sequence shown in SEQ ID NO:

90.

2. The isolated tumor-specific TCR or its antigen-binding domain as described in claim 1, wherein the amino acid sequence of the MAGE-A1 epitope is as shown in SEQ ID NO:

368.

3. The isolated tumor-specific TCR or its antigen-binding domain as described in claim 1 or 2, wherein the MHC is HLA-DRA*01:01 / DRB1*12:

02.

4. The isolated tumor-specific TCR or its antigen-binding domain as described in claim 1, comprising: The TCRα chain containing a variable region of the amino acid sequence shown in SEQ ID NO: 91 and the TCRβ chain containing a variable region of the amino acid sequence shown in SEQ ID NO:

94.

5. The isolated tumor-specific TCR or its antigen-binding domain as described in claim 1, comprising: The TCRα chain contains the amino acid sequences shown in SEQ ID NOs: 91 and 92, and the TCRβ chain contains the amino acid sequences shown in SEQ ID NOs: 94 and 95, or amino acid sequences having at least 90% sequence identity with them.

6. The isolated TCR or its antigen-binding domain according to claim 1, wherein it is isolated, purified or recombinant.

7. The isolated TCR or its antigen-binding domain according to claim 1, wherein it is chimeric, humanized, or human.

8. The isolated TCR or its antigen-binding domain according to claim 1, wherein the TCR or its antigen-binding domain is single-stranded.

9. The isolated TCR or its antigen-binding domain as described in claim 8, wherein the TCR α chain further comprises an α constant (Cα) region and / or the TCR β chain further comprises a β constant (Cβ) region.

10. The isolated TCR or its antigen-binding domain according to claim 9, wherein the Cα region and the Cβ region are mouse constant regions.

11. The isolated TCR or its antigen-binding domain according to claim 10, wherein the Cα region is composed of an amino acid sequence as shown in SEQ ID NO: 106 or an amino acid sequence having at least 90% sequence identity with it, and / or the Cβ region is composed of an amino acid sequence as shown in SEQ ID NO: 109 or an amino acid sequence having at least 90% sequence identity with it.

12. The isolated TCR or its antigen-binding domain according to claim 11, comprising: A TCRα chain consisting of an amino acid sequence as shown in SEQ ID NOs: 105 and 106 or an amino acid sequence having at least 90% sequence identity with it, and a TCRβ chain consisting of an amino acid sequence as shown in SEQ ID NOs: 108 and 109 or an amino acid sequence having at least 90% sequence identity with it.

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

14. An isolated nucleic acid molecule comprising a nucleic acid sequence comprising a nucleotide sequence encoding a TCRα chain of an isolated TCR or an antigen-binding domain thereof according to any one of claims 1-13, and a nucleotide sequence encoding a TCRβ chain of an isolated TCR or an antigen-binding domain thereof according to any one of claims 1-13.

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

16. The isolated nucleic acid molecule according to claim 14 or 15, wherein the nucleotide sequence encoding the TCRα chain and the nucleotide sequence encoding the TCRβ chain are separated by a self-splitting peptide sequence.

17. The isolated nucleic acid molecule according to claim 16, wherein the self-splitting peptide is P2A, comprising an amino acid sequence as shown in SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 111, 125, 139, 153, 167, 181 or 195.

18. Engineered immune cells, wherein the engineered immune cells comprise the isolated TCR or its antigen-binding domain as described in any one of claims 1-13 or the isolated nucleic acid molecule as described in any one of claims 14-17, and the engineered immune cells are cell lines or primary cells obtained from a subject.

19. The engineered immune cell of claim 18, wherein the engineered immune cell is a human cell.

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

21. The engineered immune cells of claim 20, wherein the engineered immune cells comprise CD8+ T cells.

22. The engineered immune cells of claim 20, wherein the engineered immune cells comprise CD4+ T cells.

23. A pharmaceutical composition comprising the engineered immune cells as described in claim 21 or 22, and a pharmaceutically acceptable carrier.

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