Modified T cells for use in the treatment of bladder cancer

Abstract

The present disclosure relates to methods of treating bladder cancer and populations of modified immunoresponsive cells expressing heterologous TCRs for use in such methods.

Description

[Technical Field]

[0001] The present disclosure relates to methods of treating bladder cancer and populations of modified immunoresponsive cells expressing heterologous TCRs for use in such methods. [Background technology]

[0002] Bladder cancer is a group of cancers that arise from the tissues of the bladder. In the UK, bladder cancer is the 11th most common cancer and the 9th leading cause of cancer death. In the US, UK, and EU, there are over 190,000 new cases of urothelial / bladder cancer each year, resulting in over 50,000 deaths. Risk factors for bladder cancer include tobacco and opioid use, exposure to carcinogens such as those in the workplace, ionizing radiation, and schistosomiasis.

[0003] Bladder cancer most commonly develops between the ages of 65 and 84, and is more prevalent in men than in women. The average 5-year survival rate after diagnosis is approximately 52%. Survival rates improve if bladder cancer is diagnosed at the earliest stages (95%) but decrease if it is diagnosed only at the later stages.

[0004] Diagnoses are staged according to the TNM classification system, where T is the size and shape of the tumor, N is the presence or absence of lymph node metastasis, and M is the presence or absence of distant metastasis. The T, N, and M features are combined to determine the cancer's "stage," ranging from 0a to IVB.

[0005] Treatment for bladder cancer varies depending on the stage of the disease, particularly how deeply the tumor has invaded the bladder wall. Generally speaking, tumors that do not subsequently invade muscle can be resected (e.g., transurethrally) with or without the addition of intravesical chemotherapy or immunotherapy. Stage II-IVA tumors that invade the muscle layer are treated multimodally with more radical resection (e.g., cystectomy or cystectomy) combined with chemotherapy or chemoradiotherapy. Stage IVB tumors are locally advanced and unresectable and are usually treated with palliative radiation therapy. Platinum-based chemotherapy agents may be used for metastatic disease. Immune checkpoint inhibitors, such as PD-1 axis antagonists, can be used if chemotherapy is contraindicated or if disease progresses despite chemotherapy.

[0006] However, existing treatment options are suboptimal for patients with advanced or metastatic bladder cancer. Only about half of those treated with standard first-line cisplatin chemotherapy respond, progressing in an average of eight months. Those ineligible for cisplatin chemotherapy may be treated with alternative regimens, but response rates are 41% or lower. Those receiving checkpoint inhibitors after progression on platinum-based chemotherapy have a response rate of only about 20%, with a median progression-free survival of about two months.

[0007] Therefore, new therapies for treating, preventing, and / or delaying the progression of bladder cancer, particularly recurrent bladder cancer, newly metastatic bladder cancer, and unresectable locally advanced bladder cancer, are desirable. Summary of the Invention

[0008] The present inventors have demonstrated that the use of engineered T cells comprising a heterologous T cell receptor capable of binding to a MAGE-A4 peptide antigen is advantageous for the treatment of bladder cancer. Engineered T cells comprising a heterologous T cell receptor capable of binding to a MAGE-A4 peptide antigen may be included, for example, in first-line treatment for bladder cancer or recurrent bladder cancer. The present inventors have devised exemplary treatment regimens in light of these findings. Engineered T cells comprising a heterologous T cell receptor capable of binding to a MAGE-A4 peptide antigen may also be included in second- or third-line treatment for bladder cancer or recurrent bladder cancer.

[0009] Including T cell therapy in earlier treatment options may alter the tumor microenvironment by infiltrating the tumor and potentially enhance and sustain T cell activation through a broader immune response. This may improve the frequency, depth, and durability of responses. Furthermore, T cell therapy has the advantage of extending to healthier patients, who have a more favorable tumor microenvironment and are better able to harvest T cells. Furthermore, healthier patients may be more likely to respond to treatment and experience fewer undesirable effects, such as cytokine release syndrome (CRS) and cytopenias.

[0010] T cell therapy can be included in a combination therapy, e.g., a combination therapy that includes an additional anti-cancer therapy, such as a checkpoint inhibitor and / or chemotherapy. Including engineered T cells in the combination therapy can be advantageous because it may allow for a reduction in the dose of the checkpoint inhibitor or additional anti-cancer therapy. This may result in a reduction in CRS and / or cytopenias, particularly when the anti-cancer therapy is chemotherapy.

[0011] Accordingly, the present disclosure provides a method of treating bladder cancer in an individual, the method comprising administering to the individual a population of modified T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4.

[0012] The present disclosure also provides a population of modified T cells comprising a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4 for use in the methods of the disclosure. [Brief explanation of the drawings]

[0013] [Figure 1] Exemplary treatment regimens for locally advanced bladder cancer that has recurred after treatment with curative intent. [Figure 2] Alternative representation of an exemplary treatment regimen for locally advanced bladder cancer that has recurred after treatment with curative intent. [Figure 3] Exemplary treatment regimens for newly metastatic or unresectable locally advanced bladder cancer. [Figure 4] Another representation of an exemplary treatment regimen for newly metastatic or unresectable locally advanced bladder cancer. [Figure 5] Efficacy of ADP-A2M4CD8 in patients with advanced urothelial carcinoma. Figure 5A: Change in baseline sum of longest diameters (SLD) of target lesions in individual patients. Figure 5B: Change in baseline target SLD by week after T cell infusion. Data show change from baseline in SLD until progression or prior to surgical resection. Investigator-assessed best overall response rate was shown according to RECIST v1.1. CR: complete response. PR: partial response. SD: stable disease. RECIST: Response Evaluation Criteria in Solid Tumors. DETAILED DESCRIPTION OF THE INVENTION

[0014] It is to be understood that the various applications of the disclosed methods and products can be tailored to the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0015] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0016] general definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a TCR" includes "TCRs," reference to "an antibody" includes two or more such antibodies, etc.

[0018] In general, the term "comprising" is intended to mean including, but not limited to. For example, the phrase "a method comprising administering a population of modified T cells" should be interpreted to mean that the method includes administering such a population, but may also include additional steps, such as administering an additional therapeutic agent.

[0019] In some aspects of the disclosure, the word "comprising" is replaced with the phrase "consisting of." The term "consisting of" is intended to be restrictive. For example, the phrase "a method consisting of administering a population of modified T cells" should be interpreted to mean that the method includes the step of administering such population, and that there are no additional steps.

[0020] The terms "protein" and "polypeptide" are used interchangeably herein and are intended to refer to polymeric chains of amino acids of any length.

[0021] Typically, the term "about" is used to refer to a value that is within ±10% (such as within ±5% or ±2%) of the value that follows it.

[0022] For purposes of this disclosure, to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the first sequence to achieve optimal alignment with the second sequence). Nucleotide residues are then compared at nucleotide position. If a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, the nucleotides are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions in the reference sequence × 100).

[0023] Typically, sequence comparison is performed over the entire length of a reference sequence. For example, if a user wants to determine whether a given ("test") sequence has a certain percent identity to SEQ ID NO:X, SEQ ID NO:X would be the reference sequence. For example, to assess whether a sequence is at least 80% identical to SEQ ID NO:X (an example of a reference sequence), one skilled in the art would align the entire length of SEQ ID NO:X and determine how many positions in the test sequence are identical to SEQ ID NO:X. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO:X. If a sequence is shorter than SEQ ID NO:X, gaps or missing positions should be considered non-identical positions.

[0024] Those skilled in the art are aware of various computer programs available to determine homology or identity between two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0025] Cancer treatment methods The present disclosure provides a method of treating bladder cancer in an individual, the method comprising administering to the individual a population of engineered T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4. As described above, the inventors have demonstrated that such a method is advantageous. In particular, engineered T cells comprising a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4 may be included in a first-line treatment for bladder cancer or recurrent bladder cancer. Engineered T cells comprising a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4 may also be included in a second- or third-line treatment for bladder cancer or recurrent bladder cancer.

[0026] In the context of this disclosure, treating bladder cancer can also include preventing and / or slowing the progression of bladder cancer.

[0027] Bladder cancer in individuals The disclosed methods are for treating bladder cancer in an individual. The individual is preferably a human. The individual may also be a non-human mammal, such as a mouse, rat, rabbit, cat, dog, pig, cow, or horse.

[0028] Bladder cancer can be any type of bladder cancer. Several types of bladder cancer are known in the art. Bladder cancer can be, for example, transitional cell bladder cancer, squamous cell bladder cancer, adenocarcinoma, sarcoma, or small cell bladder cancer. Transitional cell bladder cancer is also known as urothelial bladder cancer. Bladder cancer can be, for example, urothelial carcinoma (UCC), squamous cell carcinoma of the bladder (SCC), or adenocarcinoma of the bladder. Bladder cancer can be, for example, a solid tumor.

[0029] Bladder cancer may be a cancer that expresses MAGE-A4. Expression of MAGE-A4 has been reported in bladder cancer. For example, one study demonstrated that approximately 32% of bladder cancers express MAGE-A4.

[0030] At least 1%, e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of bladder cancer cells from an individual may express MAGE-A4. The percentage of cells expressing MAGE-A4 can be determined by any means known to those skilled in the art, such as immunohistochemistry (IHC), flow cytometry, or enzyme-linked immunosorbent assay (ELISA).

[0031] MAGE-A4 expression in bladder cancer can have an intensity of 1+ or greater (≧), for example, ≧2+ or ≧3+. Intensity scores can be assessed by IHC staining of tumors and are scored as follows: negative = no staining or staining in ≦10% or less of the stained cells; 1+ = incomplete staining in ≧10% of the stained cells; 2+ = weak to moderate staining in ≧10% of the stained cells; strong and complete staining in ≧10% of the stained cells.

[0032] The bladder cancer may be, for example, recurrent or recurrent, unresectable, or metastatic bladder cancer. For example, the bladder cancer may be unsuitable for treatment with surgical resection or radiation therapy. The bladder cancer may be, for example, ineligible for treatment with platinum-containing chemotherapy. The bladder cancer may be, for example, cisplatin-ineligible or carboplatin-ineligible.

[0033] The bladder cancer may be, for example, recurrent bladder cancer. The recurrent bladder cancer may be a locally advanced recurrence or a metastatic recurrence. The bladder cancer may be, for example, a recurrence after treatment with curative intent, such as treatment with curative intent for locally advanced bladder cancer. Thus, the individual may be a bladder cancer patient who has received treatment with curative intent, such as treatment with curative intent for locally advanced bladder cancer. Such cancer patients form a subset of bladder cancer patients that is well recognized in the art. Curative intent treatment is described in more detail below. The methods of the present disclosure may be a first-line treatment for recurrent bladder cancer.

[0034] Alternatively, the bladder cancer may be, for example, a bladder cancer that has not been previously treated. That is, the bladder cancer may be a bladder cancer that has occurred for the first time in the individual. Thus, the individual may not have been treated for bladder cancer prior to the method of the present disclosure. The bladder cancer may be, for example, newly metastatic bladder cancer or unresectable locally advanced bladder cancer. The bladder cancer may be, for example, newly metastatic bladder cancer or unresectable locally advanced bladder cancer that has not been previously treated. Thus, the individual may not have been treated for newly metastatic bladder cancer or unresectable locally advanced bladder cancer prior to the method of the present disclosure. In either case, the method of the present disclosure may be intended to treat bladder cancer that has not been previously treated. Thus, the method of the present disclosure may be a first-line treatment for bladder cancer.

[0035] In the context of the present disclosure, "selection" of a treatment may refer to a treatment regimen for a cancer that has failed a curative treatment or for which a curative treatment may be inappropriate. Curative treatment is described in more detail herein. Failure of a curative treatment may refer, for example, to the failure of a curative treatment to eliminate or induce remission of the cancer. For example, failure of a curative treatment may result in recurrence or spread of the cancer, such as local invasion or metastasis. When the cancer progresses, for example, when the cancer is locally advanced or metastatic, a curative treatment may be inappropriate. Thus, "selection" of a treatment may refer to a treatment regimen for a locally advanced cancer, a metastatic cancer, or a recurrent cancer. "Selection" of a treatment may refer to a treatment regimen for a recurrent or spread cancer.

[0036] A "choice" of treatment may be, for example, a first-line treatment for cancer. In other words, the treatment regimen may be an initial treatment regimen adopted for cancer after failure or after treatment with curative intent. For example, the treatment regimen may be an initial treatment regimen adopted for cancer after relapse or spread of cancer. For example, the treatment regimen may be an initial treatment regimen adopted for cancer after local progression / invasion, metastasis, or recurrence. A "choice" of treatment may be, for example, a second-line treatment for cancer. In other words, the treatment regimen may be, for example, a second treatment regimen adopted for cancer after failure of the first treatment regimen. Failure of the first treatment regimen may result in, for example, relapse or further spread of cancer. Failure of the first treatment regimen may result in, for example, local progression / invasion, metastasis, or recurrence. A choice of treatment may be, for example, a third-line treatment for cancer. In other words, the treatment regimen may be, for example, a third treatment regimen adopted for cancer after failure of the second treatment regimen. Failure of a second treatment regimen may result, for example, in recurrence or further spread of the cancer.Failure of a second treatment regimen may result, for example, in local progression / invasion, metastasis, or recurrence.

[0037] Treatment with curative intent Curative treatment may refer to any treatment that has the potential to cure an individual's cancer. Curative treatment may refer to, for example, a therapy administered to an individual to attempt to cure bladder cancer. Curative treatment may be administered before "selecting" treatment for relapsed or spread cancer. Thus, the cancer may have relapsed or spread after curative treatment. The cancer may have locally invaded, metastasized, or recurred after curative treatment.

[0038] The curative treatment may be any known or unknown treatment for bladder cancer. As described above, known treatments for bladder cancer include surgical resection, radiation therapy, and systemic therapy (e.g., chemotherapy). Therefore, the curative treatment may include (i) surgical resection, (ii) radiation therapy, and / or (iii) systemic therapy. For example, the curative treatment may include (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii). In certain embodiments of the present disclosure, the curative treatment may include (i); (ii); or (i) and (ii).

[0039] Systemic therapy may include or consist of (a) chemotherapy. Systemic therapy may include or consist of (b) immunotherapy. Systemic therapy may include or consist of (c) targeted therapy. For example, systemic therapy may include or consist of (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b), and (c).

[0040] Chemotherapy, such as that for bladder cancer, is well known in the art. Such chemotherapy may include, for example, platinum-based antineoplastic agents, such as cisplatin or carboplatin. Such chemotherapy may include, for example, antimetabolites, such as fluorouracil (5-FU), gemcitabine, or methotrexate. Such chemotherapy may include, for example, taxanes, such as docetaxel or paclitaxel. Such chemotherapy may include, for example, anthracyclines, such as doxorubicin. Such chemotherapy may include, for example, vinca alkaloids, such as vinblastine. Such chemotherapy may include, for example, antitumor antibiotics, such as mitomycin. Such chemotherapy may include, for example, alkylating agents, such as ifosfamide.

[0041] Immunotherapies, such as those for bladder cancer, are well known in the art. Such immunotherapies may include, for example, therapeutic immune cells, immunomodulators, checkpoint inhibitors, and vaccines. Therapeutic immune cells may include T cells, such as genetically engineered T cells, such as CAR T cells or T cells expressing a genetically engineered TCR. Immunomodulators may include, for example, interleukins, cytokines, chemokines, and immunomodulatory drugs. Immunomodulators may include, for example, multikine (leukocyte interleukin, injectable). Immunomodulators may include, for example, monalizumab, a humanized anti-NKG2A blocking antibody that prevents the inhibition of CD8+ T cells and NK cells by HLA-E-expressing tumor cells. Checkpoint inhibitors may include, for example, CTLA-4 inhibitors or PD-1 axis-binding antagonists. PD-1 axis-binding antagonists may include, for example, pembrolizumab, nivolumab, avelumab, and atezolizumab. Checkpoint inhibitors and PD-1 axis binding antagonists are described in more detail below. Vaccines can include, for example, bidutolimod (CMP-001), a toll-like receptor 9 (TLR9) agonist cancer vaccine.

[0042] Targeted therapies, such as those for bladder cancer, are well known in the art. The term targeted therapy is a term used in the art to refer to treatments that target specific genes and proteins that aid in the survival and growth of cancer cells. Targeted therapies can include, for example, fibroblast growth factor receptor inhibitors (such as erdafitinib), antibody-drug conjugates (such as enfortumab vedotin-ejfv or sacituzumab govitecan). Enfortumab vedotin-ejfv is a conjugate of a nectin-4-directed antibody and a microtubule inhibitor. Sacituzumab govitecan is a conjugate of a Trop-2-directed antibody and a topoisomerase inhibitor.

[0043] The systemic therapy may include a drug or drug combination already described for the treatment of bladder cancer, such as locally advanced or metastatic bladder cancer.The systemic therapy may include a drug or drug combination already described as a "standard of care" or approved treatment for bladder cancer, such as locally advanced or metastatic bladder cancer.

[0044] The systemic therapy may include, for example, a drug (or combination of drugs) already described for first-line treatment of bladder cancer, such as locally advanced or metastatic bladder cancer. The systemic therapy may include a drug or combination of drugs already described as a "standard of care" first-line treatment for bladder cancer, such as locally advanced or metastatic bladder cancer. For example, the systemic therapy may include cisplatin, carboplatin, gemcitabine, methotrexate, paclitaxel, doxorubicin, vinblastine, ifosfamide, pembrolizumab, avelumab, and / or atezolizumab. The systemic therapy may include, for example, monotherapy with atezolizumab, pembrolizumab, or gemcitabine. Systemic therapy may include, for example, gemcitabine and cisplatin followed by avelumab maintenance therapy; methotrexate, vinblastine, doxorubicin, cisplatin with growth factor support (ddMVAC) followed by avelumab maintenance therapy; gemcitabine and carboplatin followed by avelumab maintenance therapy; gemcitabine and paclitaxel; or combination therapy with ifosfamide, doxorubicin, and gemcitabine.

[0045] The systemic therapy may include a drug (or drug combination) already described for second-line treatment of bladder cancer (e.g., locally advanced or metastatic bladder cancer) after chemotherapy. The systemic therapy may include a drug or drug combination already described as a "standard of care" second-line treatment for bladder cancer (e.g., locally advanced or metastatic bladder cancer) after chemotherapy. For example, the systemic therapy may include pembrolizumab, paclitaxel, docetaxel, gemcitabine, nivolumab, avelumab, erdafitinib, enfortumab vedotin-ejfv, ifosfamide, doxorubicin, gemcitabine, cisplatin, methotrexate, and / or vinblastine. Systemic therapy can include, for example, monotherapy with pembrolizumab, paclitaxel, docetaxel, gemcitabine, nivolumab, avelumab, erdafitinib, or enfortumab vedotin-ejfv. Systemic therapy can include, for example, combination therapy with ifosfamide, doxorubicin, and gemcitabine; gemcitabine and paclitaxel; gemcitabine and cisplatin; or methotrexate, vinblastine, doxorubicin, cisplatin with growth factor support (ddMVAC).

[0046] The systemic therapy may include a drug (or drug combination) already described for second-line treatment of bladder cancer (e.g., locally advanced or metastatic bladder cancer) after checkpoint inhibitor therapy. The systemic therapy may include a drug or drug combination already described as a "standard of care" second-line treatment for bladder cancer (e.g., locally advanced or metastatic bladder cancer) after checkpoint inhibitor therapy. For example, the systemic therapy may include enfortumab vedotin-ejfv, gemcitabine, carboplatin, erdafitinib, paclitaxel, docetaxel, cisplatin, methotrexate, vinblastine, doxorubicin, and / or ifosfamide. The systemic therapy may include, for example, monotherapy with erdafitinib, paclitaxel, docetaxel, or gemcitabine. Systemic therapy can include, for example, combination therapy with gemcitabine and carboplatin; gemcitabine and cisplatin; methotrexate, vinblastine, doxorubicin, cisplatin with growth factor support (ddMVAC); ifosfamide, doxorubicin, and gemcitabine; or gemcitabine and paclitaxel.

[0047] Systemic therapy can include, for example, a drug (or drug combination) already described for subsequent line treatment of bladder cancer (e.g., locally advanced or metastatic bladder cancer). Systemic therapy can include a drug or drug combination already described as subsequent line treatment of the "standard of care" for bladder cancer (e.g., locally advanced or metastatic bladder cancer). The term "subsequent line" can refer to treatment after the second line. For example, the term "subsequent line" can refer to treatment that is third or fourth line. For example, systemic therapy can include enfortumab vedotin-ejfv, erdafitinib, sacituzumab govitecan-hziy, gemcitabine, paclitaxel, docetaxel, ifosfamide, doxorubicin, cisplatin, methotrexate, and / or vinblastine. Systemic therapy can include, for example, monotherapy with enfortumab vedotin-ejfv, erdafitinib, sacituzumab govitecan-hziy, gemcitabine, paclitaxel, or docetaxel. Systemic therapy can include, for example, combination therapy with ifosfamide, doxorubicin, and gemcitabine; gemcitabine and paclitaxel; gemcitabine and cisplatin; or methotrexate, vinblastine, doxorubicin, cisplatin with growth factor support (ddMVAC).

[0048] Systemic therapy may include, for example, a drug (or combination of drugs) previously described for radiosensitizing chemotherapy of bladder cancer (such as locally advanced or metastatic bladder cancer). For example, systemic therapy may include 5-FU, mitomycin, cisplatin, paclitaxel, and / or gemcitabine (e.g., low-dose gemcitabine). Systemic therapy may include, for example, monotherapy with cisplatin or gemcitabine (e.g., low-dose gemcitabine). Systemic therapy may include, for example, combination therapy with 5-FU and mitomycin; cisplatin and 5-FU; or cisplatin and paclitaxel.

[0049] The systemic therapy may include, for example, a drug (or combination of drugs) already described for the treatment of bladder cancer (such as locally advanced or metastatic bladder cancer) in patients who are ineligible for any platinum-containing chemotherapy. For example, the systemic therapy may include pembrolizumab.

[0050] In one aspect of the present disclosure, the curative treatment does not include therapeutic T cells, and thus the individual may not have received therapeutic T cells prior to the method of the present disclosure.

[0051] In either case, a cancer patient who has undergone treatment with curative intent for bladder cancer (e.g., locally advanced bladder cancer) may have their cancer essentially cured by the curative intent treatment. However, bladder cancer (e.g., locally advanced bladder cancer) may be prone to recurrence. For example, the cancer may be prone to recurrence or spread. The cancer may be prone to local invasion or metastasis. The disclosed methods may be aimed at treating recurrence, recurrence, spread, local invasion, or metastasis. The disclosed methods may be first-line treatment for recurrence, recurrence, spread, local invasion, or metastasis.

[0052] However, first-line treatment for recurrence may be a later-line (second-line, third-line, fourth-line, etc.) treatment for bladder cancer as a whole. The term "second-line treatment" may refer to a treatment regimen administered after failure of one previous line of treatment. The term "third-line treatment" may refer to a treatment regimen administered after failure of two previous line of treatments. The term "fourth-line treatment" may refer to a treatment regimen administered after failure of three previous line of treatments. In either case, remission may be partial or, preferably, complete. Any previous treatment regimen may include (i) surgical resection, (ii) radiation therapy, and / or (iii) chemotherapy. For example, the previous treatment regimen may include (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii). Failure of a previous line of treatment may result, for example, in recurrence or further spread of cancer. Failure of prior treatment options may result in, for example, local progression / invasion, metastasis, or recurrence.

[0053] Population of modified T cells The method comprises administering to an individual a population of modified T cells comprising a xenogeneic TCR capable of binding to a peptide antigen of MAGE-A4. The T cells in the population may further comprise a xenogeneic CD8 co-receptor. It is the presence of the xenogeneic TCR (and xenogeneic CD8 co-receptor, if present) that "modifies" the T cells. The xenogeneic TCR (and xenogeneic CD8 co-receptor, if present) is typically found on the surface of the modified T cells. In other words, the modified T cells may express the xenogeneic TCR (and xenogeneic CD8 co-receptor, if present) on their surface.

[0054] In the context of the present disclosure, the term "heterologous" refers to a polypeptide or nucleic acid that is foreign to a particular biological system (such as a T cell), i.e., that does not naturally occur in that system. A "heterologous" polypeptide or nucleic acid can be introduced into a system by artificial or recombinant means. Thus, heterologous expression of a TCR can alter the specificity of a T cell. Heterologous expression of a CD8 co-receptor can confer CD8 co-receptor-associated functions to a T cell. Heterologous CD8 co-receptors and heterologous TCRs are described in more detail below.

[0055] The modified T cells can include CD4+ T cells. That is, the modified T cells can include T cells that express an endogenous CD4 co-receptor. The modified T cells can include CD8+ T cells. That is, the modified T cells can include T cells that express an endogenous CD8 co-receptor. The modified T cells can include CD4+ T cells and CD8+ T cells. That is, the modified T cells can include T cells that express an endogenous CD4 co-receptor and can include T cells that express an endogenous CD8 co-receptor. Both the CD4+ T cells and the CD8+ T cells can carry a heterologous CD8 co-receptor.

[0056] The modified T cells may be allogeneic to the individual. The modified T cells may preferably be autologous to the individual. In this case, the modified T cells can be generated by modifying endogenous cells obtained from the individual. Thus, the method can include generating a population. Methods for generating modified T cells are known in the art and are discussed in the Examples below. Typically, the modified T cells of the present disclosure are generated from peripheral blood mononuclear cells (PBMCs). T cells are typically sorted from harvested cells and engineered to contain the desired modification (here, a heterologous TCR and, optionally, a heterologous CD8 co-receptor). Thus, the method can include (a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual; (b) sorting T cells from the PBMCs; and (c) generating a population by modifying the selected T cells to express a heterologous TCR and, optionally, a heterologous CD8 co-receptor.

[0057] Autologous modified T cells may be generated in anticipation of an individual's need. That is, the autologous modified T cells may be generated in advance, before the individual requires treatment with the modified cells. This can help ensure that autologous modified T cells are available for administration as soon as possible after it is determined that the individual requires treatment. In this way, the individual does not have to wait for autologous T cells to be generated before treatment can begin. This can improve the outcome of the treatment.

[0058] The proactive generation of autologous T cells may be particularly relevant in the treatment of bladder cancer at high risk of recurrence. The risk of recurrence in individuals who have undergone curative-intent treatment for bladder cancer, such as locally advanced bladder cancer, can be determined by methods routinely performed in the art. Such methods may include monitoring clinical signs or symptoms. Such methods may include, for example, one or more magnetic resonance imaging (MRI), positron emission tomography (PET), and / or computed tomography (CT) scans performed after curative-intent treatment to monitor tumor progression and / or recurrence. MRI, PET, and / or CT scans may be performed, for example, approximately every three months (e.g., once every four to sixteen weeks or once every eight to twelve weeks). If an individual is identified as at high risk of recurrence, PBMCs may be obtained at this time (i.e., before recurrence) for the purpose of generating autologous modified T cells ready for administration at the time of recurrence. Thus, when the cancer is recurrent bladder cancer, the method of the present disclosure may include: (a) obtaining peripheral blood mononuclear cells (PBMCs) from the individual; (b) sorting T cells from the PBMCs; and (c) generating a population by modifying the sorted T cells to express a xenogeneic TCR and, optionally, a xenogeneic CD8 co-receptor, wherein one or more of steps (a)-(c) are performed before recurrence. Preferably, step (a) is performed before recurrence. More preferably, steps (a) and (b) are performed before recurrence. Most preferably, steps (a), (b), and (c) are performed before recurrence. Any of these options are effective in providing treatment one step ahead at the time of recurrence.

[0059] In either case, the population of modified T cells may be administered, for example, as a single dose. The population of modified T cells may be administered, for example, as a single dose, as soon as possible after a diagnosis of bladder cancer. For example, the population of modified T cells may be administered as soon as possible after a recurrence of bladder cancer is confirmed. For example, the population of modified T cells may be administered as soon as possible after a diagnosis of previously untreated bladder cancer is confirmed.

[0060] In the context of the present disclosure, the term "as soon as possible" can refer to the earliest time point at which it is practical to administer the population of modified T cells. As explained above, generation of autologous modified T cells can take some time, so there may be a lag between the diagnosis of bladder cancer (or recurrence of bladder cancer) and the administration of treatment. Thus, administering as soon as possible after diagnosis can refer to administering as soon as practical after the autologous modified T cells are generated. Administration as soon as possible after diagnosis can refer, for example, to administration from less than about 150 days after diagnosis of bladder cancer, e.g., less than about 125 days, less than about 100 days, less than about 90 days, less than about 80 days, less than about 70 days, less than about 60 days, less than about 50 days, less than about 40 days, or less than about 30 days after diagnosis of bladder cancer. The population can be administered to the individual, for example, about 30 to about 150 days after diagnosis of bladder cancer, e.g., about 40 to about 125 days, about 50 to about 100 days, about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, or about 60 days after diagnosis of bladder cancer.

[0061] Heterogeneous TCR The engineered T cells comprise a heterologous TCR capable of binding to a peptide antigen of MAGE-A4. In other words, the engineered T cells express or display, for example, on their surface, a heterologous TCR capable of binding to a peptide antigen of MAGE-A4. MAGE-A4 is a well-known cancer antigen whose expression is restricted in normal (i.e., non-cancerous) tissues. MAGE-A4 has been shown to suppress p53 targets (such as BAX and CDKN1A) and is a binding partner of the oncogene gankyrin.

[0062] The heterologous TCR is capable of binding to a peptide antigen of MAGE-A4. For example, the heterologous TCR can bind to GVYDGREHTV (SEQ ID NO: 1), a peptide sequence known as MAGE-A4 230-239 contained in MAGE-A4. The heterologous TCR can bind to, for example, a peptide antigen of MAGE-A4 (e.g., GVYDGREHTV (SEQ ID NO: 1)) and an HLA-A molecule (e.g., HLA-A * 02 or HLA-A * A heterologous TCR can bind to a complex containing an HLA molecule, such as an HLA molecule (HLA-0201 molecule). In either case, the binding can be specific. Specificity refers to the strength of binding between a heterologous TCR and its target antigen. Specificity can be expressed by the dissociation constant Kd, which is the ratio of the bound state to the unbound state of the receptor-ligand system. Typically, the fewer different antigens a heterologous TCR can bind to other than MAGE-A4, the higher its binding specificity.

[0063] The xenogeneic TCR can bind, for example, to a MAGE-A4 peptide antigen (e.g., SEQ ID NO: 1) or a complex comprising a MAGE-A4 peptide antigen (e.g., SEQ ID NO: 1) and an HLA molecule with a dissociation constant (Kd) of 0.01 μM to 100 μM, 0.01 μM to 50 μM, 0.01 μM to 20 μM, 10 μM to 1000 μM, 10 μM to 500 μM, or 50 μM to 500 μM. For example, in a preferred embodiment of the present disclosure, the xenogeneic TCR binds to a MAGE-A4 peptide antigen or a complex comprising a MAGE-A4 peptide antigen and an HLA molecule with a Kd of 0.05 μM to 20.0 μM. For example, a heterologous TCR may be expressed at 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95μΜ, 1.0μΜ, 1.5μΜ, 2.0μΜ, 2.5μΜ, 3.0μΜ, 3.5μΜ, 4.0μΜ, 4.5μΜ, 5. 0μΜ, 5.5μΜ, 6.0μΜ, 6.5μΜ, 7.0μΜ, 7.5μΜ, 8.0μΜ, 8.5μΜ, 9.0μΜ, 9.5μΜ, The TCR can bind with a Kd of 10.0 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, or 500 μM. The Kd can be measured, for example, using surface plasmon resonance, optionally at 25° C., optionally at pH 6.5-6.9 or 7.0-7.5. The dissociation constant Kd or koff / kon can be determined by experimentally measuring the dissociation rate constant koff and the association rate constant kon. The TCR dissociation constant can be measured using a soluble form of the TCR, wherein the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain.

[0064] The heterologous TCR may be, for example, a recombinant, synthetic, or artificial TCR. That is, the heterologous TCR may be a TCR that does not occur in nature. The heterologous TCR may be, for example, an affinity-enhanced TCR, such as a specific peptide-enhanced affinity receptor (SPEAR™) TCR.

[0065] The heterologous TCR may, for example, comprise an alpha chain variable domain having at least 80% sequence identity to the sequence of amino acid residues 22-125 of SEQ ID NO: 2. The heterologous TCR may, for example, comprise a beta chain variable domain having at least 80% sequence identity to the sequence of amino acid residues 22-123 of SEQ ID NO: 3. The heterologous TCR may, for example, comprise an alpha chain variable domain having at least 80% sequence identity to the sequence of amino acid residues 22-125 of SEQ ID NO: 2 and a beta chain variable domain having at least 80% sequence identity to the sequence of amino acid residues 22-123 of SEQ ID NO: 3. The alpha chain variable domain may, for example, have at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of amino acid residues 22-125 of SEQ ID NO: 2. The alpha chain variable domain may, for example, comprise or consist of amino acid residues 22-125 of SEQ ID NO: 2. The beta chain variable domain can have, for example, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of amino acid residues 22 to 123 of SEQ ID NO: 3. The beta chain amino acid sequence can, for example, comprise or consist of amino acid residues 22 to 123 of SEQ ID NO: 3.

[0066] The heterologous TCR may, for example, comprise an alpha chain having at least 80% sequence identity to the sequence of amino acid residues 22-282 of SEQ ID NO: 2. The heterologous TCR may, for example, comprise a beta chain having at least 80% sequence identity to the sequence of amino acid residues 22-311 of SEQ ID NO: 3. The heterologous TCR may, for example, comprise an alpha chain having at least 80% sequence identity to the sequence of amino acid residues 22-282 of SEQ ID NO: 2 and a beta chain variable domain having at least 80% sequence identity to the sequence of amino acid residues 22-311 of SEQ ID NO: 3. The alpha chain variable domain may, for example, have at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of amino acid residues 22-282 of SEQ ID NO: 2. The alpha chain variable domain may, for example, comprise or consist of amino acid residues 22-282 of SEQ ID NO: 2. The beta chain variable domain can have, for example, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of amino acid residues 22 to 311 of SEQ ID NO: 3. The beta chain amino acid sequence can, for example, comprise or consist of amino acid residues 22 to 311 of SEQ ID NO: 3.

[0067] Heterologous TCRs are typically expressed with an N-terminal signal peptide that is cleaved prior to expression at the surface of a T cell. In this regard, amino acids 1-21 of SEQ ID NO:2 and SEQ ID NO:3, respectively, are typically cleaved prior to expression of the TCR at the surface of a T cell. A heterologous TCR may, for example, comprise an alpha chain amino acid sequence having at least 80% sequence identity to SEQ ID NO:2. A heterologous TCR may, for example, comprise a beta chain amino acid sequence having at least 80% sequence identity to SEQ ID NO:3. A heterologous TCR may, for example, comprise an alpha chain amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 and a beta chain amino acid sequence having at least 80% sequence identity to SEQ ID NO:3. The alpha chain amino acid sequence may, for example, have at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2. The alpha chain amino acid sequence may, for example, comprise or consist of SEQ ID NO:2. The beta chain amino acid sequence can have, for example, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. The beta chain amino acid sequence can, for example, comprise or consist of SEQ ID NO: 3.

[0068] Heterologous TCRs can be, for example, (i) an alpha chain variable domain comprising a CDR1 comprising (1) the sequence of SEQ ID NO:4 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO:4; (ii) an alpha chain variable domain comprising a CDR2 comprising (1) the sequence of SEQ ID NO:5 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO:5; or (iii) an alpha chain variable domain comprising a CDR3 comprising (1) the sequence of SEQ ID NO:6 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO:6. (iv) a beta chain variable domain comprising a CDR1 comprising (1) the sequence of SEQ ID NO:7 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO:7; (v) a beta chain variable domain comprising a CDR2 comprising (1) the sequence of SEQ ID NO:8 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO:8; and / or (vi) a beta chain variable domain comprising a CDR3 comprising (1) the sequence of SEQ ID NO:9 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO:9.

[0069] The alpha chain of a heterologous TCR may comprise, for example, (i) an alpha chain variable domain comprising a CDR1 comprising (1) the sequence of SEQ ID NO: 4, or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO: 4; (ii) an alpha chain variable domain comprising a CDR2 comprising (1) the sequence of SEQ ID NO: 5, or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO: 5; and (iii) an alpha chain variable domain comprising a CDR3 comprising (1) the sequence of SEQ ID NO: 6, or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO: 6. The alpha chain of a heterologous TCR may comprise, for example, (i) an alpha chain variable domain comprising a CDR1 comprising the sequence of SEQ ID NO: 4; (ii) an alpha chain variable domain comprising a CDR2 comprising the sequence of SEQ ID NO: 5; and (iii) an alpha chain variable domain comprising a CDR3 comprising the sequence of SEQ ID NO: 6.

[0070] The beta chain of a heterologous TCR may comprise, for example, (vi) a beta chain variable domain comprising a CDR1 comprising (1) the sequence of SEQ ID NO: 7 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO: 7; (v) a beta chain variable domain comprising a CDR2 comprising (1) the sequence of SEQ ID NO: 8 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO: 8; and (vi) a beta chain variable domain comprising a CDR3 comprising (1) the sequence of SEQ ID NO: 9 or (2) an amino acid sequence comprising an insertion, deletion, or substitution of one, two, or three amino acids relative to the sequence of SEQ ID NO: 9. The beta chain of a heterologous TCR may comprise, for example, (iv) a beta chain variable domain comprising a CDR1 comprising the sequence of SEQ ID NO: 7; (v) a beta chain variable domain comprising a CDR2 comprising the sequence of SEQ ID NO: 8; and (vi) a beta chain variable domain comprising a CDR3 comprising the sequence of SEQ ID NO: 9.

[0071] The heterologous TCR may, for example, comprise an alpha chain comprising a CDR1 having the sequence of SEQ ID NO: 4, a CDR2 having the sequence of SEQ ID NO: 5, and a CDR3 having the sequence of SEQ ID NO: 6, and a beta chain comprising a CDR1 having the sequence of SEQ ID NO: 7, a CDR2 having the sequence of SEQ ID NO: 8, and a CDR3 having the sequence of SEQ ID NO: 9. The heterologous TCR may further comprise, for example, any of the percent identities of the alpha and beta chains discussed herein.

[0072] Heterologous CD8 co-receptor The modified T cells can comprise a heterologous CD8 co-receptor, in other words, the modified T cells can express, for example, a heterologous CD8 co-receptor on their surface.

[0073] CD8 is a cell surface glycoprotein naturally found on most cytotoxic T lymphocytes and mediates efficient cell-cell interactions within the immune system. CD8 acts as a co-receptor for the T cell receptor, resulting in the recognition of antigens presented by antigen-presenting cells in the context of class I MHC molecules. The CD8 co-receptor binds to class I MHC and enhances TCR signaling. Functional co-receptors can be homodimers consisting of two CD8 alpha chains or heterodimers consisting of one CD8 alpha chain and one CD8 beta chain.

[0074] Thus, the heterologous CD8 co-receptor comprised in the engineered T cell may be CD8α. In other words, the heterologous CD8 co-receptor may be a homodimer consisting of two CD8 alpha chains. Alternatively, the heterologous CD8 co-receptor may be a heterodimer consisting of one CD8 alpha chain and one CD8 beta chain. In either case, the CD8 alpha chain may comprise or consist of an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 10. Thus, the heterologous CD8 co-receptor may comprise an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 10.

[0075] Both the CD8 alpha and beta chains share high homology with immunoglobulin variable light chains. The CD8 alpha and beta chains have CDR-like loops involved in MHC-class I binding. A heterologous CD8 coreceptor can comprise, for example, a CD8 alpha chain comprising: (i) an alpha chain CDR1 comprising (1) the sequence of SEQ ID NO: 11 or (2) an amino acid sequence containing one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO: 11; (ii) an alpha chain CDR2 comprising (1) the sequence of SEQ ID NO: 12 or (2) an amino acid sequence containing one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO: 12; and / or (iii) an alpha chain CDR3 comprising (1) the sequence of SEQ ID NO: 13 or (2) an amino acid sequence containing one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO: 13.

[0076] The heterologous CD8 co-receptor is capable of binding to a class I MHC molecule. The heterologous CD8 co-receptor can bind, for example, to the α3 portion of a class I MHC molecule, e.g., via an IgV-like domain of the CD8 co-receptor. The α3 portion is typically found between residues 223 and 229 of a class I MHC molecule. The ability of the heterologous CD8 co-receptor to bind to a class I MHC molecule improves the ability of the modified T cell to engage with a cognate antigen via the heterologous TCR. The cognate antigen, MAGE-A4, typically binds to HLA-A. * The heterologous CD8 co-receptor is presented as a complex with class I MHC molecules such as 02. The off-rate (k off ) can be improved or increased. This improvement or increase can be for engineered T cells that contain a heterologous TCR that binds to MAGE-A4 but lack a heterologous CD8 co-receptor. The heterologous CD8 co-receptor can, for example, help organize the heterologous TCR on the surface of the engineered cell, thereby improving the ability of the heterologous TCR to participate in TCR / peptide-MHCI interactions. The heterologous CD8 co-receptor can, for example, bind or interact with LCK (lymphocyte-specific protein tyrosine kinase) in a zinc-dependent manner and activate transcription factors such as NFAT, NF-κB, and AP-1. Thus, expression of a heterologous CD8 co-receptor can improve the affinity and / or avidity of the engineered T cells for MAGE-A4 and / or improve activation upon binding to MAGE-A4. Methods for determining affinity, avidity, and T cell activation are well known in the art. Expression of a heterologous CD8 co-receptor can improve or increase, for example, the expression of CD40L, cytokine production, cytotoxic activity, induction of dendritic cell maturation, or induction of dendritic cell cytokine production of the engineered T cells in response to antigen (MAGE-A4) binding, relative to engineered T cells that contain a heterologous TCR that binds to a peptide antigen of MAGE-A4 but lack a heterologous CD8 co-receptor.

[0077] CD8α and HLA-A *Synergy has been demonstrated between the heterologous CD8 co-receptor and the peptide antigen presented on HLA-A. Thus, in one embodiment of the present disclosure, the heterologous CD8 co-receptor may be CD8α and the heterologous TCR may be HLA-A. * It may be capable of binding to a peptide antigen of MAGE-A4 complexed with 0201. The peptide antigen may be, for example, SEQ ID NO:1.

[0078] Checkpoint inhibitors The method may include administering a checkpoint inhibitor to the individual. Thus, the method may include administering to the individual (i) a population of engineered T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4 and (ii) a checkpoint inhibitor. In other words, the method may include combination treatment with (i) a population of engineered T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4 and (ii) a checkpoint inhibitor. As described below, the combination may also include an additional anti-cancer therapy, such as chemotherapy.

[0079] The checkpoint inhibitor may be administered to an individual in the same selection of treatment as the population of modified T cells. As noted above, a "selection" of treatment may refer to a particular treatment regimen for a cancer that has failed a curative treatment or for which a curative treatment may be inappropriate. Thus, a checkpoint inhibitor and a population of modified T cells may be administered as part of the same treatment regimen for a cancer that has failed a curative treatment or for which a curative treatment may be inappropriate. The checkpoint inhibitor and the population of modified T cells may be administered as part of a first-line treatment regimen. The checkpoint inhibitor and the population of modified T cells may be administered as part of a second-line treatment regimen. A second-line treatment regimen may be employed, for example, after failure of a first-line treatment regimen. The checkpoint inhibitor and the population of modified T cells may be administered as part of a third-line treatment regimen. A third-line treatment regimen may be employed, for example, after failure of a second-line treatment regimen.

[0080] Checkpoint inhibitor therapy is a form of cancer immunotherapy. This therapy targets immune checkpoints, which are important regulators of the immune system that, when stimulated, can dampen the immune response to immunological stimuli. Checkpoint therapy can block inhibitory checkpoints and restore immune system function. Checkpoint inhibitors can target the molecules CTLA4, PD-1, and PD-L1.

[0081] Checkpoint inhibitors can target, for example, CTLA4. That is, checkpoint inhibitors can include CTLA4 blockers. CTLA4 blockers are known in the art and include, for example, ipilimumab.

[0082] Checkpoint inhibitors may include PD-1 axis-binding antagonists. Administration of PD-1 axis-binding antagonists (e.g., pembrolizumab) has been described in the art as a first-line "standard of care" treatment for bladder cancer, such as locally advanced or metastatic bladder cancer. For example, monotherapy with pembrolizumab or nivolumab has been described as a first-line "standard of care" treatment for locally advanced or metastatic bladder cancer. Monotherapy with pembrolizumab has also been described as a second-line "standard of care" treatment for locally advanced or metastatic bladder cancer after chemotherapy. Pembrolizumab has also been described in the art as a treatment for bladder cancer (such as locally advanced or metastatic bladder cancer) in patients who are ineligible for any platinum-containing chemotherapy.

[0083] Programmed cell death protein 1 (PD-1, also known as CD279) is a protein expressed on the surface of T cells that regulates immune responses by maintaining T cell homeostasis. Ligation of PD-1 with one of its ligands (PD-L1 or PD-L2) mediates inhibitory signals within T cells. Specifically, PD-1-generated signals block phosphorylation of key TCR signaling intermediates, thereby terminating early TCR signaling and reducing T cell activation. This results in a reduction in T cell effector functions (e.g., proliferation, cytotoxicity, and cytokine production) and an impaired ability to transition to memory T cells.

[0084] PD-L1 and PD-L2 are members of the B7 family. PD-L1 protein is upregulated on certain activated immune cells (e.g., macrophages, dendritic cells, T cells, and B cells) and is also expressed in certain normal tissues. PD-L1 is also highly expressed in many cancers. PD-L2 is expressed primarily on dendritic cells and in some tumors. Because many cancers express PD-1 ligands, the PD-1 axis has an established role in cancer immune evasion and tumor resistance.

[0085] When PD-1 ligands are expressed by cancers, such as solid tumors, the tumor microenvironment becomes immunosuppressive. This may inhibit the function of engineered T cells that infiltrate the tumor. Endogenous anti-tumor T cell responses may also be inhibited. Thus, tumors are more likely to evade the immune system. In the present disclosure, PD-1 axis-binding antagonists can be administered to negate the suppressive effects of PD-L1 and / or PD-L2 expression in the tumor microenvironment. By negating suppression, the function of engineered and / or endogenous T cells can be sustained. That is, administration of a PD-1 axis-binding antagonist can sustain the function of the engineered T cells and / or their progeny included in the administration. Administration of a PD-1 axis-binding antagonist can sustain the function of endogenous T cells in an individual. Administration of a PD-1 axis-binding antagonist can sustain the function of the engineered T cells (and / or their progeny) included in the administration and endogenous T cells in the individual. In either case, endogenous T cells may be included in, for example, the tumor microenvironment. For example, the endogenous T cells may be tumor-infiltrating lymphocytes (TILs).

[0086] Sustained T cell function can refer, for example, to maintaining, restoring, and / or enhancing T cell function. Sustained T cell function can refer, for example, to sustaining T cell activation. In this way, the duration of an effective T cell response can be extended. In other words, sustained activation can be associated with improved duration of effector function (such as cytokine production, cytotoxicity, and / or proliferation). Sustained activation can also support the ability of T cells to transition to memory T cells. The generation of memory T cells is advantageous because it can enable anti-tumor immunity to be maintained for a long period of time, for example, for several months or years. Methods for determining activation, cytokine production, cytotoxicity, proliferation, and the generation of memory T cells are well known in the art.

[0087] Administration of PD-1 axis-binding antagonists can sustain the function of engineered T cells and / or endogenous T cells by reducing exhaustion. Exhaustion can be reduced within a population of engineered T cells and / or within T cells that are progeny of the population of engineered T cells. Exhaustion can be reduced within endogenous T cells in an individual. (i) Exhaustion can be reduced within a population of engineered T cells and / or within T cells that are progeny of the population of engineered T cells, and (ii) within endogenous T cells in an individual. Exhausted T cells typically express high levels of PD-1 and experience loss of function. For example, exhausted T cells may have a reduced ability to produce cytokines such as IL-2 or TNFα. Exhausted T cells may have a reduced proliferative capacity. Exhausted T cells may have reduced cytotoxic activity. Ultimately, exhausted T cells can be targeted for destruction. Thus, exhaustion leads to loss of T cell function or even the loss of T cells, which is detrimental to tumor immunity. Therefore, reducing T cell exhaustion can improve treatment outcomes.

[0088] In the context of the present disclosure, a PD-1 axis binding antagonist is a molecule that inhibits the interaction between PD-1 and a PD-1 ligand and / or the transmission of a signal resulting from the interaction between PD-1 and a PD-1 ligand. The PD-1 ligand can be PD-L1 or PD-L2. A PD-1 axis binding antagonist can, for example, reduce or block the interaction between PD-1 and a PD-1 ligand. A PD-1 axis binding antagonist can, for example, reduce or block the transmission of a signal resulting from the interaction between PD-1 and a PD-1 ligand. A PD-1 axis binding antagonist can block, inhibit, or reduce the biological activity of PD-1 and / or a PD-1 ligand.

[0089] By inhibiting the interaction between PD-1 and its ligand and / or the transmission of signals resulting from the interaction between PD-1 and its ligand, PD-1 axis-binding antagonists can sustain (e.g., maintain, restore, or enhance) the function of endogenous T cells. Similarly, PD-1 axis-binding antagonists can sustain (e.g., maintain, restore, or enhance) the function of engineered T cells administered to an individual. Sustained function can be demonstrated, for example, by the maintenance or improvement of T cell proliferation, cytokine production, target cell killing, activation, CD28 signaling, ability to infiltrate tumors, ability to recognize and bind antigens presented by dendritic cells, and / or ability to produce interferon. In this way, PD-1 axis-binding antagonists negate the immunosuppressive properties of the tumor microenvironment.

[0090] The PD-1 axis binding antagonist can be, for example, a PD-1 binding antagonist. That is, the PD-1 axis binding antagonist can inhibit (e.g., block or reduce) the binding of PD-1 to its binding partner. For example, the PD-1 axis binding antagonist can inhibit the binding of PD-1 to PD-L1, PD-L2, or both PD-L1 and PD-L2. The PD-1 binding antagonist can be, for example, an antibody that binds to PD-1, or an antigen-binding variant or fragment thereof. The PD-1 binding antagonist can be, for example, an antibody that binds to SEQ ID NO: 14, or an antigen-binding variant or fragment thereof. Antibodies that bind to PD-1 are known in the art and include, for example, nivolumab, pembrolizumab, cemiplimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, retifanlimab (INCMGA00012), AMP-224, MEDI0680 (AMP-514), sasanlimab, budigalimab, ezabenlimab (BI 754091), and zimberelimab (AB122). Thus, the PD-1 axis-binding antagonist may be nivolumab, pembrolizumab, cemiplimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, retifanlimab (INCMGA00012), AMP-224, MEDI0680 (AMP-514), sasanlimab, budigalimab, ezabenlimab (BI 754091), or zimberelimab (AB122), or any combination thereof. The PD-1 axis-binding antagonist may be, for example, pembrolizumab. Alternatively, the PD-1 binding antagonist may be any immunoadhesin, protein, or oligopeptide that inhibits, reduces, blocks, or interferes with signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2.

[0091] The heavy and light chain sequences of nivolumab are set forth in SEQ ID NOs: 17 and 18, respectively. The heavy and light chain sequences of pembrolizumab are set forth in SEQ ID NOs: 19 and 20, respectively. The heavy and light chain sequences of cemiplimab are set forth in SEQ ID NOs: 21 and 22, respectively. The heavy and light chain sequences of dostallimab are set forth in SEQ ID NOs: 31 and 32, respectively. Armed with knowledge of the heavy and light chain sequences of a given antibody, one of skill in the art can identify antigen-binding variants or fragments of the antibody using methods routinely performed in the art. An antigen-binding variant or fragment of nivolumab may, for example, comprise the three CDRs contained in SEQ ID NO: 17 and the three CDRs contained in SEQ ID NO: 18. An antigen-binding variant or fragment of pembrolizumab may, for example, comprise the three CDRs contained in SEQ ID NO: 19 and the three CDRs contained in SEQ ID NO: 20. An antigen-binding variant or fragment of cemiplimab may, for example, comprise the three CDRs contained in SEQ ID NO: 21 and the three CDRs contained in SEQ ID NO: 22. An antigen-binding variant or fragment of dostarlimab may, for example, comprise the three CDRs contained in SEQ ID NO: 31 and the three CDRs contained in SEQ ID NO: 32. Methods for identifying CDRs within a heavy or light chain sequence are routine in the art.

[0092] The PD-1 axis-binding antagonist may be, for example, a PD-L1 binding antagonist. That is, the PD-1 axis-binding antagonist can inhibit (e.g., block or reduce) the binding of PD-L1 to a binding partner. For example, the PD-1 axis-binding antagonist can inhibit the binding of PD-L1 to PD-1. The PD-L1 binding antagonist may be, for example, an antibody that binds to PD-L1 or an antigen-binding variant or fragment thereof. The PD-L1 binding antagonist may be, for example, an antibody that binds to SEQ ID NO: 15 or an antigen-binding variant or fragment thereof. Antibodies that bind to PD-L1 are known in the art and include, for example, durvalumab, atezolizumab, avelumab, BMS 936559 (MDX-1105), envafolimab (KN035), and cosibelimab (CK-301). Thus, the PD-L1 axis-binding antagonist can be durvalumab, atezolizumab, avelumab, BMS 936559 (MDX-1105), embafolimab (KN035), or cosibelimab (CK-301), or any combination thereof. Alternatively, the PD-L1 axis-binding antagonist can be any immunoadhesin, protein, or oligopeptide that inhibits, reduces, blocks, or interferes with signaling resulting from the interaction of PD-L1 with PD-1.

[0093] The heavy and light chain sequences of durvalumab are set forth in SEQ ID NOs: 23 and 24, respectively. The heavy and light chain sequences of atezolizumab are set forth in SEQ ID NOs: 25 and 26, respectively. The heavy and light chain sequences of avelumab are set forth in SEQ ID NOs: 27 and 28, respectively. The heavy and light chain sequences of BMS 936559 (MDX-1105) are set forth in SEQ ID NOs: 29 and 30, respectively. Armed with knowledge of the heavy and light chain sequences of a given antibody, one of skill in the art can identify antigen-binding variants or fragments of the antibody using methods routinely performed in the art. An antigen-binding variant or fragment of durvalumab may, for example, comprise the three CDRs contained in SEQ ID NO: 23 and the three CDRs contained in SEQ ID NO: 24. An antigen-binding variant or fragment of atezolizumab may, for example, comprise the three CDRs contained in SEQ ID NO: 25 and the three CDRs contained in SEQ ID NO: 26. An antigen-binding variant or fragment of avelumab may, for example, comprise the three CDRs contained in SEQ ID NO: 27 and the three CDRs contained in SEQ ID NO: 28. An antigen-binding variant or fragment of BMS 936559 (MDX-1105) may, for example, comprise the three CDRs contained in SEQ ID NO: 29 and the three CDRs contained in SEQ ID NO: 30. Methods for identifying CDRs within a heavy or light chain sequence are routine in the art.

[0094] The PD-1 axis-binding antagonist may be, for example, a PD-L2 binding antagonist. That is, the PD-1 axis-binding antagonist can inhibit (e.g., block or reduce) the binding of PD-L2 to a binding partner. The PD-L2 binding antagonist may be, for example, an antibody that binds to PD-L2, or an antigen-binding variant or fragment thereof. The PD-L2 binding antagonist may be, for example, an antibody that binds to SEQ ID NO: 16, or an antigen-binding variant or fragment thereof. Alternatively, the PD-L2 binding antagonist may be any immunoadhesin, protein, or oligopeptide that inhibits, reduces, prevents, or interferes with signaling resulting from the interaction of PD-L2 with PD-1.

[0095] When the PD-1 axis-binding antagonist is an antibody (such as a known antibody or its antigen-binding variant), it may be, for example, a monoclonal antibody, a human or humanized antibody, a full-length antibody, a diabody, a linear antibody, or a single-chain antibody molecule. The antibody isotype can be selected from any of the five immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated alpha, delta, epsilon, gamma, and mu (M), respectively. Antibodies of the gamma and alpha classes may be any of the subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. When the PD-1 axis-binding antagonist is an antigen-binding fragment of an antibody, it may be, for example, an Fv, Fab, Fab', Fab'-SH, F(ab')2, or scFv.

[0096] When the PD-1 axis binding antagonist is an immunoadhesin, the immunoadhesin can comprise an adhesin domain that confers binding activity for a PD-1 axis component (e.g., PD-1, PD-L1, or PD-L2) and an immunoglobulin constant domain. The immunoglobulin constant domain can be of any isotype, such as IgG1, IgG2, IgG2A, IgG2B, IgG3, IgG4 subtypes, IgA, IgA1, IgA2, IgE, IgD, or IgM. The immunoglobulin constant domain can comprise, for example, (i) the hinge, CH2, and CH3 regions or (ii) the hinge, CH1, CH2, and CH3 regions of an immunoglobulin molecule. Thus, the immunoadhesin may comprise (a) the extracellular or PD-1-binding portion of PD-L1 or PD-L2, or the extracellular or PD-L1- or PD-L2-binding portion of PD-1 fused to (b) a constant domain of an immunoglobulin sequence.

[0097] At least 1% of bladder cancer cells from an individual may express PD-L1, e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. Bladder cancer cells from an individual may express PD-L1 with a tumor proportion score (TPS) of 1% or more (≧), e.g., ≧2%, ≧10%, or ≧50%. The proportion of cells expressing PD-L1 can be determined by any means known to one of skill in the art, such as IHC, flow cytometry, or ELISA.

[0098] At least 1%, e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of bladder cancer cells from an individual may express PD-L2. Bladder cancer from an individual may express PD-L2 with a tumor proportion score (TPS) of 1% or more (≧), e.g., ≧2%, ≧10%, or ≧50%. The proportion of cells expressing PD-L2 can be determined by any means known to one of skill in the art, such as IHC, flow cytometry, or ELISA.

[0099] Expression of PD-1, PD-L1, and / or PD-L2 in bladder cancer can have an intensity of 1+ or greater (≧), for example, ≧2+ or ≧3+. Intensity scores can be assessed by IHC staining of tumors and are scored as follows: negative = no staining or staining in ≦10% of stained cells; 1+ = incomplete staining in ≧10% of stained cells; 2+ = weak to moderate staining in ≧10% of stained cells; strong and complete staining in ≧10% of stained cells.

[0100] The population of engineered T cells and the checkpoint inhibitor may be administered in any order and at any number of times. The population of engineered T cells may be administered, for example, as a single dose. The checkpoint inhibitor can be administered, for example, (a) before the engineered T cells, (b) simultaneously with the engineered T cells, and / or (c) after the engineered T cells. For example, the checkpoint inhibitor may be administered, for example, (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b), and (c). When the checkpoint inhibitor is administered simultaneously with the population of engineered T cells, the checkpoint inhibitor and the population of engineered T cells may be included in the same composition or in separate compositions.

[0101] Administering a checkpoint inhibitor simultaneously with the engineered T cells can refer to administering the checkpoint inhibitor and the engineered T cells substantially simultaneously. In other words, the dose of the checkpoint inhibitor may be administered at about the same time as the dose of the population of engineered T cells. For example, the dose of the checkpoint inhibitor may be administered within about 2 hours, within about 4 hours, within about 6 hours, within about 8 hours, within about 12 hours, or within about 24 hours of the dose of the population of engineered T cells.

[0102] Administering a checkpoint inhibitor prior to the engineered T cells can refer to administering the checkpoint inhibitor at any time prior to the engineered T cells. In other words, the dose of the checkpoint inhibitor can be administered at any time prior to the dose of the population of engineered T cells. For example, the dose of the checkpoint inhibitor can be administered about 24 hours or more, about 36 hours or more, about 48 hours or more, about 72 hours or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 2 weeks or more, about 3 weeks or more, or about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, about 8 weeks or more, or about 12 weeks or more prior to the population of engineered T cells. The checkpoint inhibitor can also be administered prior to the engineered T cells, for example, to initiate treatment while generating autologous engineered T cells.

[0103] Administering a checkpoint inhibitor after the engineered T cells can refer to administering the checkpoint inhibitor at any time after the engineered T cells. In other words, the dose of the checkpoint inhibitor can be administered at any time after the dose of the population of engineered T cells. For example, the dose of the checkpoint inhibitor can be administered about 24 hours or more, about 36 hours or more, about 48 hours or more, about 72 hours or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 2 weeks or more, about 3 weeks or more, or about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, about 8 weeks or more, or about 12 weeks or more after the population of engineered T cells.

[0104] If the checkpoint inhibitor is administered before the population of engineered T cells, the checkpoint inhibitor may be continued after administration of the engineered T cells. Thus, in one embodiment of the present disclosure, administration of the checkpoint inhibitor is initiated before administration of the population of engineered T cells and continued after administration of the population of engineered T cells. In other words, a dose of the checkpoint inhibitor may be administered before the dose of the population of engineered T cells, and one or more additional doses of the checkpoint inhibitor may be administered thereafter. The dose of the checkpoint inhibitor may be administered, for example, according to known therapeutic regimens for checkpoint inhibitors. The purpose of the additional doses of the checkpoint inhibitor may be to maintain the effect achieved by administration of the initial dose. Each of the one or more additional doses may contain the same checkpoint inhibitor as the initial dose, or may contain a different checkpoint inhibitor than the initial dose.

[0105] When the checkpoint inhibitor is administered simultaneously with the population of engineered T cells, the checkpoint inhibitor may be continued after administration of the engineered T cells. Thus, in one embodiment of the present disclosure, administration of the checkpoint inhibitor is initiated simultaneously with administration of the population of engineered T cells and continues after administration of the population of engineered T cells. In other words, a dose of the checkpoint inhibitor may be administered simultaneously with a dose of the population of engineered T cells, and one or more additional doses of the checkpoint inhibitor may be administered later. The dose of the checkpoint inhibitor may be administered, for example, according to a known therapeutic regimen for the checkpoint inhibitor. The purpose of the additional dose of the checkpoint inhibitor may be to maintain the effect achieved by administration of the initial dose. Each of the one or more additional doses may include the same checkpoint inhibitor as the initial dose, or may include a different checkpoint inhibitor than the initial dose.

[0106] If the checkpoint inhibitor is administered after the population of engineered T cells, the checkpoint inhibitor may be continued after the initial administration. Thus, in one embodiment of the present disclosure, administration of the checkpoint inhibitor begins after administration of the population of engineered T cells and continues after the initial administration of the checkpoint inhibitor. In other words, a dose of the checkpoint inhibitor may be administered after the dose of the population of engineered T cells, and one or more additional doses of the checkpoint inhibitor may be administered later. The dose of the checkpoint inhibitor may be administered, for example, according to a known therapeutic regimen for the checkpoint inhibitor. The purpose of the additional dose of the checkpoint inhibitor may be to maintain the effect achieved by administration of the initial dose. Each of the one or more additional doses may contain the same checkpoint inhibitor as the initial dose, or may contain a different checkpoint inhibitor than the initial dose.

[0107] In either case, one or more additional doses of the checkpoint inhibitor may be administered at any suitable interval. Suitable dosing intervals for checkpoint inhibitors are known in the art and may be specific to the identity of the checkpoint inhibitor. For example, in the case of a PD-1 axis binding antagonist such as pembrolizumab, one or more additional doses may be administered, for example, once about every two weeks (Q2W) starting two weeks after administration of the first dose. One or more additional doses may be administered, for example, once about every three weeks (Q3W) starting three weeks after administration of the first dose. One or more additional doses may be administered, for example, once about every four weeks (Q4W) starting four weeks after administration of the first dose. One or more additional doses may be administered, for example, once about every five weeks (Q5W) starting five weeks after administration of the first dose. One or more additional doses may be administered, for example, once about every six weeks (Q6W) starting six weeks after administration of the first dose. In preferred embodiments of the present disclosure, the one or more additional doses are administered about once every three weeks (Q3W) starting three weeks after administration of the first dose, or about once every six weeks (Q6W) starting six weeks after administration of the first dose.

[0108] Any number of additional doses of checkpoint inhibitor may be administered, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more additional doses. Additional doses may be administered until disease progression, unacceptable toxicity, withdrawal of consent, or death.

[0109] Additional anticancer therapy The method may include administering to the individual an additional anti-cancer therapy. Thus, the method may include administering to the individual (i) a population of modified T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4 and (ii) an additional anti-cancer therapy. In other words, the method may include combination treatment of the individual with (i) a population of modified T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4 and (ii) an additional anti-cancer therapy. A checkpoint inhibitor, such as a PD-1 axis binding antagonist, may be included in the combination.

[0110] The additional anti-cancer therapy may be administered to the individual in the same selection of treatment as the population of modified T cells. As noted above, a "selection" of treatment may refer to a particular treatment regimen for a cancer that has failed a curative treatment or for which a curative treatment may be inappropriate. Thus, the additional anti-cancer therapy and the population of modified T cells may be administered as part of the same treatment regimen for a cancer that has failed a curative treatment or for which a curative treatment may be inappropriate. The additional anti-cancer therapy and the population of modified T cells may be administered as part of a first-line treatment regimen. The additional anti-cancer therapy and the population of modified T cells may be administered as part of a second-line treatment regimen. The second-line treatment regimen may be employed, for example, after the failure of a first-line treatment regimen. The additional anti-cancer therapy and the population of modified T cells may be administered as part of a third-line treatment regimen. The third-line treatment regimen may be employed, for example, after the failure of a second-line treatment regimen.

[0111] The individual may be administered at least one additional anti-cancer therapy, for example, one or more, two or more, three or more, four or more, or five or more additional anti-cancer therapies.

[0112] The additional anti-cancer therapy may be, for example, an anti-cancer drug therapy. In other words, the additional anti-cancer therapy may be a systemic therapy. The additional anti-cancer therapy may, for example, include or consist of (a) chemotherapy. The additional anti-cancer therapy may, for example, include or consist of (b) immunotherapy. The additional anti-cancer therapy may, for example, include or consist of (c) targeted therapy. For example, the additional anti-cancer therapy may include or consist of (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b), and (c). In either case, the chemotherapy may be systemic chemotherapy or intravesical chemotherapy. Intravesical chemotherapy is chemotherapy administered locally to the bladder.

[0113] Optional chemotherapy may be included in the additional anti-cancer therapy. The method may include administering one or more chemotherapeutic agents (e.g., two or more, three or more, four or more, or five or more chemotherapeutic agents). The method may include administering a platinum-based anti-neoplastic agent, such as cisplatin or carboplatin. The method may include administering an antimetabolite, such as fluorouracil (5-FU), gemcitabine, or methotrexate. The method may include administering a taxane, such as docetaxel or paclitaxel. The method may include administering an anthracycline, such as doxorubicin. The method may include administering a vinca alkaloid, such as vinblastine. The method may include administering an antitumor antibiotic, such as mitomycin. The method may include administering an alkylating agent, such as ifosfamide.

[0114] Any immunotherapy may be included in the additional anticancer therapy. The method may include administering one or more immunotherapies (e.g., two or more, three or more, four or more, or five or more immunotherapies). Immunotherapies, such as those for bladder cancer, are well known in the art. Such immunotherapies may include, for example, therapeutic immune cells, immunomodulators, checkpoint inhibitors, and vaccines. Therapeutic immune cells may include T cells, e.g., engineered T cells, such as CAR T cells or T cells expressing an engineered TCR. Immunomodulators may include, for example, interleukins, cytokines, chemokines, and immunomodulatory agents. Immunomodulators may include, for example, multikine (leukocyte interleukin, injectable). Immunomodulators may include, for example, monalizumab, a humanized anti-NKG2A blocking antibody that prevents the inhibition of CD8+ T cells and NK cells by HLA-E-expressing tumor cells. Checkpoint inhibitors may include, for example, CTLA-4 inhibitors or PD-1 axis binding antagonists. PD-1 axis binding antagonists can include, for example, pembrolizumab, nivolumab, avelumab, and atezolizumab. Checkpoint inhibitors and PD-1 axis binding antagonists are described in more detail below. Vaccines can include, for example, bidutolimod (CMP-001), a toll-like receptor 9 (TLR9) agonist cancer vaccine.

[0115] Any targeted therapy may be included in the additional anticancer therapy. The method may include administering one or more targeted therapies (e.g., two or more, three or more, four or more, or five or more targeted therapies). Targeted therapies, such as those for bladder cancer, are well known in the art. The method may include administering, for example, an inhibitor of a fibroblast growth factor receptor, such as erdafitinib. The method may include administering, for example, an antibody-drug conjugate, such as enfortumab vedotin-ejfv or sacituzumab govitecan. Enfortumab vedotin-ejfv is a conjugate of a nectin-4-directed antibody and a microtubule inhibitor. Sacituzumab govitecan is a conjugate of a Trop-2-directed antibody and a topoisomerase inhibitor.

[0116] The additional anti-cancer therapy may include a drug or drug combination already described for the treatment of bladder cancer, such as locally advanced or metastatic bladder cancer. The additional anti-cancer therapy may include a drug or drug combination already described as an approved treatment or "standard of care" for bladder cancer, such as locally advanced or metastatic bladder cancer. In this context, approval may relate to approval by, for example, the FDA, EMA, or MHRA.

[0117] The additional anticancer therapy may include, for example, a drug (or drug combination) already described for first-line treatment of bladder cancer, such as locally advanced or metastatic bladder cancer. The additional anticancer therapy may include a drug or drug combination already described as a "standard of care" first-line treatment for bladder cancer, such as locally advanced or metastatic bladder cancer. For example, the additional anticancer therapy may include cisplatin, carboplatin, gemcitabine, methotrexate, paclitaxel, doxorubicin, vinblastine, ifosfamide, pembrolizumab, avelumab, and / or atezolizumab. The additional anticancer therapy may include, for example, monotherapy with atezolizumab, pembrolizumab, or gemcitabine. Additional anticancer therapy may include, for example, gemcitabine and cisplatin followed by avelumab maintenance therapy; methotrexate, vinblastine, doxorubicin, cisplatin with growth factor support (ddMVAC) followed by avelumab maintenance therapy; gemcitabine and carboplatin followed by avelumab maintenance therapy; gemcitabine and paclitaxel; or combination therapy with ifosfamide, doxorubicin, and gemcitabine.

[0118] The additional anticancer therapy may include, for example, a drug (or drug combination) already described for second-line treatment of bladder cancer (such as locally advanced or metastatic bladder cancer) after chemotherapy. The additional anticancer therapy may include a drug or drug combination already described as a "standard of care" second-line treatment for bladder cancer (such as locally advanced or metastatic bladder cancer) after chemotherapy. For example, the additional anticancer therapy may include pembrolizumab, paclitaxel, docetaxel, gemcitabine, nivolumab, avelumab, erdafitinib, enfortumab vedotin-ejfv, ifosfamide, doxorubicin, gemcitabine, cisplatin, methotrexate, and / or vinblastine. Additional anticancer therapy may include, for example, monotherapy with pembrolizumab, paclitaxel, docetaxel, gemcitabine, nivolumab, avelumab, erdafitinib, or enfortumab vedotin-ejfv. Additional anticancer therapy may include, for example, combination therapy with ifosfamide, doxorubicin, and gemcitabine; gemcitabine and paclitaxel; gemcitabine and cisplatin; or methotrexate, vinblastine, doxorubicin, cisplatin (ddMVAC) with growth factor support. Second-line treatment may also be considered first-line treatment for recurrent or relapsed cancer.

[0119] The additional anticancer therapy may include, for example, a drug (or drug combination) already described for second-line treatment of bladder cancer (e.g., locally advanced or metastatic bladder cancer) after checkpoint inhibitor therapy. The additional anticancer therapy may include a drug or drug combination already described as a "standard of care" second-line treatment for bladder cancer (e.g., locally advanced or metastatic bladder cancer) after checkpoint inhibitor therapy. For example, the additional anticancer therapy may include enfortumab vedotin-ejfv, gemcitabine, carboplatin, erdafitinib, paclitaxel, docetaxel, cisplatin, methotrexate, vinblastine, doxorubicin, and / or ifosfamide. The additional anticancer therapy may include, for example, monotherapy with erdafitinib, paclitaxel, docetaxel, or gemcitabine. Additional anticancer therapies may include, for example, combination therapy with gemcitabine and carboplatin; gemcitabine and cisplatin; methotrexate, vinblastine, doxorubicin, cisplatin with growth factor support (ddMVAC); ifosfamide, doxorubicin, and gemcitabine; or gemcitabine and paclitaxel. Second-line treatment may also be considered first-line treatment for recurrent or relapsed cancer.

[0120] The additional anticancer therapy may include, for example, a drug (or drug combination) already described for subsequent line treatment of bladder cancer (e.g., locally advanced or metastatic bladder cancer). The additional anticancer therapy may include a drug or drug combination already described as subsequent line treatment of "standard of care" for bladder cancer (e.g., locally advanced or metastatic bladder cancer). For example, the additional anticancer therapy may include enfortumab vedotin-ejfv, erdafitinib, sacituzumab govitecan-hziy, gemcitabine, paclitaxel, docetaxel, ifosfamide, doxorubicin, cisplatin, methotrexate, and / or vinblastine. The additional anticancer therapy may include, for example, monotherapy with enfortumab vedotin-ejfv, erdafitinib, sacituzumab govitecan-hziy, gemcitabine, paclitaxel, or docetaxel. Additional anti-cancer therapy may include, for example, ifosfamide, doxorubicin, and gemcitabine; gemcitabine and paclitaxel; gemcitabine and cisplatin; or combination therapy with methotrexate, vinblastine, doxorubicin, cisplatin (ddMVAC) with growth factor support.

[0121] The additional anticancer therapy may include, for example, a drug (or drug combination) already described for radiosensitizing chemotherapy of bladder cancer (such as locally advanced or metastatic bladder cancer). For example, the additional anticancer therapy may include 5-FU, mitomycin, cisplatin, paclitaxel, and / or gemcitabine (e.g., low-dose gemcitabine). The additional anticancer therapy may include, for example, monotherapy with cisplatin or gemcitabine (e.g., low-dose gemcitabine). The additional anticancer therapy may include, for example, combination therapy with 5-FU and mitomycin; cisplatin and 5-FU; or cisplatin and paclitaxel.

[0122] The additional anti-cancer therapy may include, for example, a drug (or combination of drugs) already described for the treatment of bladder cancer (such as locally advanced or metastatic bladder cancer) in patients who are ineligible for any platinum-containing chemotherapy. For example, the additional anti-cancer therapy may include pembrolizumab.

[0123] The population of engineered T cells and the one or more additional anti-cancer therapies may be administered in any order and at any number of times. As described above, the population of engineered T cells may be administered, for example, as a single dose. The additional anti-cancer therapy can be administered, for example, (a) before the engineered T cells, (b) simultaneously with the engineered T cells, and / or (c) after the engineered T cells. For example, the additional anti-cancer therapy may be administered, for example, (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b), and (c). When the additional anti-cancer therapy is administered simultaneously with the population of engineered T cells, the additional anti-cancer therapy and the population of engineered T cells may be included in the same composition or in separate compositions.

[0124] Administration of an additional anti-cancer therapy simultaneously with the engineered T cells can refer to administering the additional anti-cancer therapy and the engineered T cells substantially simultaneously. In other words, the dose of the additional anti-cancer therapy can be administered approximately simultaneously with the dose of the population of engineered T cells. For example, the dose of the additional anti-cancer therapy can be administered within about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, or about 24 hours of the dose of the population of engineered T cells.

[0125] Administration of an additional anti-cancer therapy prior to the engineered T cells can refer to administration of the additional anti-cancer therapy at any time prior to the engineered T cells. In other words, the dose of the additional anti-cancer therapy can be administered at any time prior to the dose of the population of engineered T cells. For example, the dose of the additional anti-cancer therapy can be administered about 24 hours or more, about 36 hours or more, about 48 hours or more, about 72 hours or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 2 weeks or more, about 3 weeks or more, or about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, about 8 weeks or more, or about 12 weeks or more prior to the population of engineered T cells. The additional anti-cancer therapy can also be administered prior to the engineered T cells, for example, to initiate treatment while generating autologous engineered T cells.

[0126] Administration of an additional anti-cancer therapy after the engineered T cells can refer to administration of the anti-cancer therapy at any time after the engineered T cells. In other words, the dose of the additional anti-cancer therapy can be administered at any time after the dose of the population of engineered T cells. For example, the dose of the additional anti-cancer therapy can be administered about 24 hours or more, about 36 hours or more, about 48 hours or more, about 72 hours or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 2 weeks or more, about 3 weeks or more, or about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, about 8 weeks or more, or about 12 weeks or more after the population of engineered T cells. The additional anti-cancer therapy can be administered after the engineered T cells, for example, to initiate treatment while generating autologous engineered T cells.

[0127] If the additional anticancer therapy is administered before the population of modified T cells, the additional anticancer therapy may be continued after the administration of the modified T cells. Thus, in one embodiment of the present disclosure, the administration of the additional anticancer therapy is initiated before the administration of the population of modified T cells and continued after the administration of the population of modified T cells. In other words, a dose of the additional anticancer therapy may be administered before the dose of the population of modified T cells, and one or more additional doses of the additional anticancer therapy may be administered afterwards. The dose of the additional anticancer therapy may be administered, for example, according to a known treatment regimen of the additional anticancer therapy. The purpose of the additional dose of the additional anticancer therapy may be to maintain the effect achieved by the administration of the initial dose. Each of the one or more additional doses may include the same additional anticancer therapy as the initial dose, or may include an additional anticancer therapy different from the initial dose.

[0128] When an additional anti-cancer therapy is administered simultaneously with the population of modified T cells, the additional anti-cancer therapy may be continued after administration of the modified T cells. Thus, in one embodiment of the present disclosure, administration of the additional anti-cancer therapy is initiated simultaneously with administration of the population of modified T cells and continues after administration of the population of modified T cells. In other words, a dose of the additional anti-cancer therapy may be administered simultaneously with a dose of the population of modified T cells, and one or more additional doses of the additional anti-cancer therapy may be administered later. The dose of the additional anti-cancer therapy may be administered, for example, according to a known treatment regimen of the additional anti-cancer therapy. The purpose of the additional dose of the additional anti-cancer therapy may be to maintain the effect achieved by administration of the initial dose. Each of the one or more additional doses may include the same additional anti-cancer therapy as the initial dose, or may include an additional anti-cancer therapy different from the initial dose.

[0129] When an additional anticancer therapy is administered after the population of modified T cells, the additional anticancer therapy may be continued after the initial administration. Thus, in one embodiment of the present disclosure, the administration of the additional anticancer therapy begins after the administration of the population of modified T cells and continues after the initial administration of the additional anticancer therapy. In other words, a dose of the additional anticancer therapy may be administered after the dose of the population of modified T cells, and one or more additional doses of the additional anticancer therapy may be administered later. The dose of the additional anticancer therapy may be administered, for example, according to a known treatment regimen of the additional anticancer therapy. The purpose of the additional dose of the additional anticancer therapy may be to maintain the effect achieved by the administration of the initial dose. Each of the one or more additional doses may include the same additional anticancer therapy as the initial dose, or may include an additional anticancer therapy different from the initial dose.

[0130] In either case, the one or more further doses of the additional anti-cancer therapy may be administered at any suitable interval. Suitable dosing intervals for additional anti-cancer therapies are known in the art and may be specific to the identity of the additional anti-cancer therapy.

[0131] Any number of additional doses of additional anti-cancer therapy may be administered, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more additional doses. Additional doses may be administered until disease progression, unacceptable toxicity, withdrawal of consent, or death.

[0132] Combination Treatment Protocol Examples of combination treatment protocols are shown in Figures 1-4. Figures 1 and 2 relate to the treatment of bladder cancer that has recurred after treatment with curative intent, e.g., locally advanced bladder cancer. This treatment is a first-line treatment. In this case, a checkpoint inhibitor (such as a PD-1 axis binding inhibitor, e.g., pembrolizumab) may be administered simultaneously or substantially simultaneously with the modified T cells. In other words, a dose of the checkpoint inhibitor may be administered approximately simultaneously with a dose of the population of modified T cells. For example, a dose of the checkpoint inhibitor may be administered within about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, or about 24 hours of a dose of the population of modified T cells. Alternatively, the checkpoint inhibitor may be administered after the modified T cells. Administration of the checkpoint inhibitor may continue, for example, after administration of the modified T cells. In other words, a dose of the checkpoint inhibitor may be administered approximately simultaneously with a dose of the population of modified T cells, and one or more additional doses of the checkpoint inhibitor may be administered thereafter. The dose of the checkpoint inhibitor may be administered according to known treatment regimens for checkpoint inhibitors, for example, treatment regimens for PD-1 axis binding inhibitors (such as pembrolizumab) are described above.

[0133] In Figures 1 and 2, administration of the modified T cells and checkpoint inhibitor can be a second-line or subsequent-line (e.g., third-line or fourth-line) treatment for bladder cancer overall. In Figures 1 and 2, administration of the modified T cells and checkpoint inhibitor can be a first-line treatment for recurrence.

[0134] As an alternative to or in addition to the checkpoint inhibitors in Figures 1 and 2, additional anti-cancer therapy, such as chemotherapy, may be administered.

[0135] In Figures 1 and 2, cells (e.g., PBMCs) for generating the population of modified T cells are obtained from an individual prior to relapse. For example, the cells may be obtained when the individual is identified as being at high risk for relapse. This allows for generation of the population of modified T cells to begin (and ideally be completed) before the cancer recurs. In turn, this may allow for a shorter period between relapse and administration of the population of modified T cells, i.e., administering the population of modified T cells as soon as possible after diagnosis of recurrent cancer, potentially improving treatment outcomes.

[0136] 3 and 4 relate to the treatment of previously untreated bladder cancer (e.g., newly metastatic or unresectable locally advanced bladder cancer). This treatment is a first-line treatment. In this case, a checkpoint inhibitor (such as a PD-1 axis binding inhibitor, e.g., pembrolizumab) may be administered before the modified T cells. In other words, a dose of the checkpoint inhibitor may be administered before a dose of the population of modified T cells. For example, a dose of the checkpoint inhibitor may be administered about 24 hours or more, about 36 hours or more, about 48 hours or more, about 72 hours or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 2 weeks or more, about 3 weeks or more, or about 4 weeks or more before the population of modified T cells. Administration of the checkpoint inhibitor may continue, for example, after administration of the modified T cells. In other words, a dose of the checkpoint inhibitor may be administered before a dose of the population of modified T cells, and one or more additional doses of the checkpoint inhibitor may be administered thereafter. The dose of the checkpoint inhibitor may be administered, for example, according to known treatment regimens for checkpoint inhibitors. Treatment regimens for PD-1 axis binding inhibitors (e.g., pembrolizumab) are as described above.

[0137] In Figures 3 and 4, administration of engineered T cells and checkpoint inhibitors can be a first-line treatment for bladder cancer.

[0138] In Figure 2, an additional anti-cancer therapy, such as chemotherapy, may be administered instead of or in addition to the checkpoint inhibitor.

[0139] In Figures 3 and 4, once cancer is diagnosed, cells (e.g., PBMCs) for generating a population of modified T cells may be obtained from an individual. Generation of the population of modified T cells may then begin. Treatment with a checkpoint inhibitor can be administered during the generation period, and the population of modified T cells is administered when ready. This allows treatment to begin as soon as possible after cancer diagnosis, potentially improving treatment outcomes.

[0140] Administration of engineered T cells may be advantageous in combination therapies such as those discussed in Figures 1-4. In particular, administration of engineered T cells may allow for reduced doses of checkpoint inhibitors and / or additional anti-cancer therapy, potentially resulting in reduced cytokine release syndrome (CRS) and / or cytopenias, particularly when the additional anti-cancer therapy is chemotherapy.

[0141] In combination therapy, the administration of a checkpoint inhibitor and / or additional anticancer therapy may be advantageous. In particular, the checkpoint inhibitor and / or additional anticancer therapy can be administered while the autologous population of modified T cells is being generated, thereby allowing treatment to be initiated as soon as possible. This may improve treatment outcomes. The administration of the checkpoint inhibitor and / or additional anticancer therapy and the modified T cells may also have an adjuvant effect.

[0142] Administration As described above, the population of engineered T cells can be administered to an individual as soon as possible after a diagnosis of bladder cancer or recurrent bladder cancer. The population can be administered to an individual, for example, less than about 150 days after a diagnosis of bladder cancer, e.g., less than about 125 days, less than about 100 days, less than about 90 days, less than about 80 days, or less than about 70 days after a diagnosis of bladder cancer. The population can be administered to an individual, for example, about 30 to about 150 days after a diagnosis of bladder cancer, e.g., about 40 to about 125 days, about 50 to about 100 days, about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, or about 60 days after a diagnosis of bladder cancer. Typically, the population of engineered T cells is administered as a single dose. However, one or more additional doses (e.g., two or more, three or more, four or more, or five or more) can be administered depending on patient factors and the physician's discretion.

[0143] The population can include any number of modified T cells that is therapeutically effective. The number of modified T cells for a given individual can vary depending on factors such as the cancer being treated, the severity or stage of the cancer, the age of the patient, etc. Thus, the number administered can vary at the discretion of the physician and can be specific to each subject. In one example, the population can be about 0.8 x 10 9 ~About 10×10 9 modified T cells, e.g., about 0.8 x 10 9 ~Approx. 1.2×10 9 modified T cells, approximately 1.2 x 10 9 ~Approx. 6×10 9 modified T cells, or approximately 1.0 x 10 9 ~10×10 9 The population may comprise, for example, 1.0 x 10 modified T cells. 9 modified T cells, approximately 5.0 x 10 9 modified T cells, or approximately 10 x 10 9 The modified T cells may comprise:

[0144] Typically, the population of engineered T cells is administered intravenously, but any suitable route may be used, such as, for example, intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal routes.

[0145] The population of engineered T cells may be administered in combination with a checkpoint inhibitor and / or an additional anti-cancer therapy, as described above. The checkpoint inhibitor or additional anti-cancer therapy may be administered by any route appropriate for a given therapy, such as intravenously, intramuscularly, subcutaneously, intradermally, transdermally, or intraperitoneally. One or more doses of the checkpoint inhibitor or anti-cancer therapy may be administered, as described above.

[0146] Each dose of the checkpoint inhibitor or additional anti-cancer therapy can contain any therapeutically effective amount of the checkpoint inhibitor or additional anti-cancer therapy. The amount for a given individual can vary depending on factors such as the cancer being treated, the severity or stage of the cancer, the age of the patient, etc. Thus, the amount administered can vary according to the judgment of the physician and can be specific to each subject.

[0147] As an example, the checkpoint inhibitor may include a PD-1 axis-binding antagonist such as pembrolizumab. The initial dose of the PD-1 axis-binding antagonist and / or any further doses of the PD-1 axis-binding antagonist may include about 200 mg to about 700 mg of the PD-1 axis-binding antagonist, for example, about 200 mg to about 400 mg of the PD-1 axis-binding antagonist. For example, the initial dose of the PD-1 axis-binding antagonist and / or any further doses of the PD-1 axis-binding antagonist may include about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg of the PD-1 axis-binding antagonist. The initial dose of the PD-1 axis-binding antagonist and / or any further doses of the PD-1 axis-binding antagonist may include, for example, about 200 mg to about 400 mg of the PD-1 axis-binding antagonist, for example, about 480 mg of the PD-1 axis-binding antagonist. In a preferred embodiment of the disclosure, the initial dose of the PD-1 axis binding antagonist and any further doses of the PD-1 axis binding antagonist comprise 200 mg Q3W pembrolizumab or 400 mg Q6W pembrolizumab.

[0148] In some embodiments of the present disclosure, the method includes administering lymphocyte-depleting chemotherapy to the individual prior to administering the population of modified T cells. That is, lymphocyte-depleting chemotherapy may be administered prior to step (a). Lymphocyte-depleting chemotherapy may be administered, for example, from about 14 days before step (a) to about 1 day before step (a), for example, from about 13 days before step (a) to about 2 days before step (a), from about 12 days before step (a) to about 3 days before step (a), from about 11 days before step (a) to about 4 days before step (a), from about 10 days before step (a) to about 5 days before step (a), or from about 9 days before step (a) to about 1 day before step (a). The lymphocyte-depleting chemotherapy may be administered from about 7 days before step (a) to about 6 days before step (a), from about 8 days before step (a) to about 7 days before step (a), from about 10 days before step (a) to about 1 day before step (a), from about 9 days before step (a) to about 2 days before step (a), from about 8 days before step (a) to about 3 days before step (a), from about 7 days before step (a) to about 4 days before step (a), or from about 6 days before step (a) to about 5 days before step (a). Preferably, the lymphocyte-depleting chemotherapy is administered from about 7 days before step (a) to about 4 days before step (a). The purpose of lymphocyte-depleting chemotherapy may be to deplete the individual's lymphocyte compartment to provide space for the adoptively transferred modified T cells to expand. In this way, the effect of a given dose of modified T cells can be maximized. The lymphocyte-depleting chemotherapy may include any suitable lymphotoxic agent. Lymphocytotoxic agents and suitable dosages are known in the art. Lymphocyte-depleting chemotherapy may include, for example, fludarabine and / or cyclophosphamide. Typically, lymphocyte-depleting chemotherapy is administered intravenously. Any suitable route may be used, such as, for example, intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal routes.

[0149] Pharmaceutical and medical uses The present disclosure provides a population of modified T cells comprising a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4 for use in a method of treating bladder cancer in an individual. Any of the aspects described above in relation to the method of the disclosure may also be applied to the population for use.

[0150] The present disclosure also provides the use of a population of modified T cells in the manufacture of a medicament for use in a method of treating bladder cancer in an individual, wherein the modified T cells comprise a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4. Any of the aspects described above in relation to the method of the disclosure may also be applied to the use of this population.

[0151] The present disclosure also provides the use of a population of modified T cells in a method of treating bladder cancer in an individual, wherein the modified T cells comprise a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4. Any of the aspects described above in relation to the method of the present disclosure may also be applied to the use of this population. [Example]

[0152] Introduction ADP-A2M4CD8-specific peptide-enhanced affinity receptor (SPEAR™) T cells have been genetically engineered to target the tumor antigen MAGE-A4 under appropriate human leukocyte antigen (HLA) expression. ADP-A2M4CD8 are autologous CD4 and CD8 positive T cells transduced with a self-inactivating (SIN) lentiviral vector expressing a high-affinity MAGE-A4-specific T cell receptor (TCR) and an additional CD8α co-receptor.

[0153] The affinity-optimized TCR (ADP-A2M4 TCR) contains an alpha chain variable domain contained in SEQ ID NO: 2 and a beta chain variable domain contained in SEQ ID NO: 3. When expressed in T cells, the signal peptides are cleaved from SEQ ID NOs: 2 and 3 prior to surface expression. The A2M4 TCR targets the tumor antigen MAGE-A4 and activates engineered T cells. HLA-A * When presented in the O2-GVYDGREHTV antigen complex, it recognizes the MAGE-A4 230-239 (GVYDGREHTV; SEQ ID NO: 1) peptide sequence derived from MAGE-A4.

[0154] The CD8α co-receptor contained in ADP-A2M4CD8 SPEAR™ T cells is engineered to provide additional functionality to CD4+ T cells. Because CD4+ T cells have weak effector function in response to class I antigens, the CD8α co-receptor was introduced together with the TCR to increase TCR binding avidity and enhance the polyfunctional response of genetically engineered CD4+ T cells against MAGE-A4-positive tumors. Co-expression of CD8α conferred CD8+ killer T cell potential to CD4+ helper T cells while maintaining or enhancing their helper cell potential. The addition of the CD8α co-receptor directly affected TCR binding to HLA-peptide complexes in CD4+ T cells, enhancing CD4+ T cell effector function. Thus, ADP-A2M4CD8 SPEAR™ T cells were engineered to improve ADP-A2M4-expressing T cells.

[0155] This was confirmed in preclinical in vitro assays, in which ADP-A2M4CD8 showed a clear improvement in T cell activation compared with ADP-A2M4-expressing T cells (when cultured with antigen-positive cells), particularly as measured by increased CD40L surface expression in the CD4+ fraction. The inclusion of dendritic cells (DCs) in the cocultures resulted in a significant improvement in ADP-A2M4CD8 T cells. Compared to cultures containing ADP-A2M4 cells, cytokine release from both DCs (IL-12, MIG) and T cells (IFNγ, IL-2, and other Th1 cells) was improved. Furthermore, transduction with ADP-A2M4CD8 resulted in the conversion of CD4+ T cells from being unable to kill MAGE-A4-positive 3D microspheres to having effective cytotoxic function. Thus, ADP-A2M4CD8-transduced CD4+ T cells exhibit not only CD4+ helper function but also improved T cell effector function.

[0156] However, existing treatment options are suboptimal for patients with advanced or metastatic bladder cancer. Only about half of individuals treated with standard first-line cisplatin chemotherapy respond, progressing in an average of eight months. Individuals who are ineligible for cisplatin chemotherapy may be treated with alternative regimens, but response rates are below 41%. Individuals who receive checkpoint inhibitors after progression on platinum-based chemotherapy have a response rate of only about 20%, with a median progression-free survival of approximately two months.

[0157] Improved regimens for treating bladder cancer are desirable. Examples 1 and 2 discuss regimens using ADP-A2M4CD8 as first-line treatment for (1) recurrent bladder cancer and (2) newly metastatic or unresectable locally advanced bladder cancer, respectively. This regimen is based on promising results from preliminary clinical trials involving ADP-A2M4CD8 in bladder cancer. In these trials, the majority (6 of 7) of individuals with late-stage metastatic urothelial carcinoma who received ADP-A2M4CD8 monotherapy experienced shrinkage of target lesions, with two patients experiencing confirmed responses. A third individual had an unconfirmed response, and a fourth individual experienced a 62% tumor shrinkage.

[0158] Example 1. Recurrent bladder cancer. Subjects are selected for treatment with ADP-A2M4CD8. Briefly, subjects to be selected must have previously been diagnosed with locally advanced bladder cancer (particularly urothelial carcinoma) and have undergone curative treatment such as surgical resection, chemotherapy, and / or radiation therapy. Subjects to be selected typically undergo follow-up PET and / or CT scans every three months after curative treatment. These scans identify whether the subject is at high risk of recurrence. Subjects at high risk of recurrence may be selected for treatment.

[0159] Additionally, the selected subjects were HLA-A * 02:01, HLA-A * 02:03, or HLA-A *02:06, or another HLA-A with the same protein sequence in the peptide-binding domain * Positive for the 02 allele. HLA-A * 02:05 Positive patients have two HLA-A * Since alloreactivity from ADP-A2M4 and ADP-A2M4CD8 to 02:05-positive cell lines has already been confirmed, they are excluded from this study. * HLA-A as the 02 allele * 02:07 or any A * Patients with any of the O2 null alleles are also excluded because these alleles result in reduced activity.

[0160] Selected subjects also have tumors in which ≧30% of tumor cells exhibit MAGE-A4 expression defined as ≧2+ by immunohistochemistry (IHC).

[0161] Autologous cells are collected by leukapheresis from a selected subject for processing and manufacturing into ADP-A2M4CD8. The heterologous TCR contained in the ADP-A2M4CD8 T cells comprises an alpha chain sequence contained in SEQ ID NO:2 and a beta chain sequence contained in SEQ ID NO:3. The heterologous CD8 co-receptor contained in the ADP-A2M4CD8 T cells comprises two CD8 alpha chains, each comprising the amino acid sequence of SEQ ID NO:10. The surface-expressed heterologous TCR and surface-expressed heterologous CD8 co-receptor do not contain signal sequences. Preferably, the autologous cells are collected before a patient is diagnosed with recurrent, locally advanced, or metastatic bladder cancer.

[0162] A baseline tumor assessment is obtained prior to treatment. Subjects then undergo lymphodepleting chemotherapy with fludarabine and cyclophosphamide (days -7 to -4) in anticipation of receiving ADP-A2M4CD8. Subjects receive a single intravenous infusion of ADP-A2M4CD8 on day 1. Treatment with standard of care anticancer therapy (typically pembrolizimab) is also administered concomitantly (e.g., within approximately 14 days of ADP-A2M4CD8 administration).

[0163] 1×108 ~1×10 10 ADP-A2M4CD8 T cells are administered to the subject. The initial dose selected for ADP-A2M4CD8 is 1 x 10 9 transduced cells (range: 0.8 × 10 9 ~1.2×10 9 transduced cells).

[0164] Subjects will be monitored immediately after infusion (Days 1-8). Subjects will be monitored weekly until Week 4 after infusion. Subjects will then be monitored at Weeks 6, 8, 12, 16, and 24, and at least every 3 months thereafter until disease progression. Tumor response will be assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Additional data collected will include: - Core needle biopsies to directly assess the "immune landscape" within the tumor at baseline and throughout the study. - Serum cytokine levels at baseline and throughout the study period. - Humoral immune responses to tumor antigens using serum at baseline and throughout the study. - Serum antibodies against ADP-A2M4CD8 at baseline and throughout the study period. - Soluble markers of tumors and their microenvironment using liquid biopsies. For example, markers of circulating tumor cells (CTCs), exosomes, and cell-free DNA (cfDNA) produced by dying tumor cells can be used to monitor both the molecular signature of tumor burden (including target antigen expression) and immune responses. Analysis of such soluble markers allows for estimation of systemic tumor burden and genetic profiling, including MAGE-A4 mRNA expression and mutational profiling. Analysis of such soluble markers also allows for systemic assessment of immune responses. - Phenotype and activity of genetically modified T cells before and after infusion. Relevant assays can be performed on blood and tumors if resections have been performed. Assays include: (i) cell generation These include (ii) phenotypic analysis to determine T cell lineage in the blood (and tumor if resection is performed) during and after infusion; (ii) quantification of the senescence and activation state of immune subsets from PBMCs; (iii) analysis of gene expression or epigenetic profiles to reflect the phenotype and functional state of the cells; and / or (iv) direct functional assessment of the cells. - Persistence of the infused genetically engineered cells and its correlation with therapeutic efficacy. Persistence can be determined by data on the number of copies of genetically modified DNA per μg of DNA and / or the number of transduced cells or total lymphocytes per μL. Well-established methodologies include (i) quantification of ADP-A2M4CD8 cells by quantitative PCR of the transgene from DNA extracted from frozen PBMCs and (ii) quantification and phenotypic analysis of ADP-A2M4CD8 cells by flow cytometry, DNA analysis, and RNA analysis from frozen PBMCs.

[0165] Figures 1 and 2 summarize the above process.

[0166] Max 10×10 9 Doses of ADP-A2M4CD8 were administered and shown to be well tolerated. New data suggest that when first-line treatment of recurrent bladder cancer includes administration of ADP-A2M4CD8, treatment outcomes may be improved, for example, compared to "standard of care" first-line treatment for recurrent bladder cancer.

[0167] Example 2. Newly metastatic or unresectable locally advanced bladder cancer. Subjects are selected for treatment with ADP-A2M4CD8. Briefly, the subjects selected have newly metastatic or unresectable locally advanced bladder cancer (particularly urothelial carcinoma). The selected subjects also have HLA-A * 02:01, HLA-A * 02:03, or HLA-A * 02:06, or another HLA-A with the same protein sequence in the peptide-binding domain *Positive for the 02 allele. HLA-A * 02:05 Positive patients have two HLA-A * Since alloreactivity from ADP-A2M4 and ADP-A2M4CD8 to 02:05-positive cell lines has already been confirmed, they are excluded from this study. * HLA-A as the 02 allele * Patients with either the 02:07 or any A*02 null allele will also be excluded, as these alleles result in reduced activity. Additionally, selected subjects will have tumors in which ≥30% of tumor cells demonstrate MAGE-A4 expression, defined as ≥2+ by immunohistochemistry (IHC).

[0168] Autologous cells are collected by leukapheresis from enrolled subjects for processing and manufacturing into ADP-A2M4CD8. The heterologous TCR contained in the ADP-A2M4CD8 T cells comprises an alpha chain sequence contained in SEQ ID NO:2 and a beta chain sequence contained in SEQ ID NO:3. The heterologous CD8 co-receptor contained in the ADP-A2M4CD8 T cells comprises two CD8 alpha chains, each comprising the amino acid sequence of SEQ ID NO:10. The surface-expressed heterologous TCR and surface-expressed heterologous CD8 co-receptor do not contain signal sequences.

[0169] A baseline tumor assessment is obtained before treatment. Subjects then receive standard-of-care anticancer therapy, typically pembrolizimab. In preparation for ADP-A2M4CD8, subjects undergo lymphodepleting chemotherapy with fludarabine and cyclophosphamide (days -7 to -4). Subjects receive a single intravenous infusion of ADP-A2M4CD8 on day 1. Day 1 is scheduled as soon as possible after the diagnosis of newly metastatic or unresectable locally advanced bladder cancer, i.e., as soon as autologous cells are available. This is typically around 60 days after leukapheresis.

[0170] 1×10 8 ~1×10 10ADP-A2M4CD8 T cells are administered to the subject. The initial dose selected for ADP-A2M4CD8 is 1 x 10 9 transduced cells (range: 0.8 × 10 9 ~1.2×10 9 transduced cells).

[0171] Subjects will be monitored immediately after infusion (Days 1-8). Subjects will be monitored weekly until Week 4 after infusion. Subjects will then be monitored at Weeks 6, 8, 12, 16, and 24, and at least every 3 months thereafter until disease progression. Tumor response will be assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Additional data collected will include: - Core needle biopsies to directly assess the "immune landscape" within the tumor at baseline and throughout the study. - Serum cytokine levels at baseline and throughout the study period. - Humoral immune responses to tumor antigens using serum at baseline and throughout the study. - Serum antibodies against ADP-A2M4CD8 at baseline and throughout the study period. - Soluble markers of tumors and their microenvironment using liquid biopsies. For example, markers of circulating tumor cells (CTCs), exosomes, and cell-free DNA (cfDNA) produced by dying tumor cells can be used to monitor both the molecular signature of tumor burden (including target antigen expression) and immune responses. Analysis of such soluble markers allows for estimation of systemic tumor burden and genetic profiling, including MAGE-A4 mRNA expression and mutational profiling. Analysis of such soluble markers also allows for systemic assessment of immune responses. - Phenotype and activity of the genetically modified T cells before and after infusion. Relevant assays can be performed using blood and, if resection was performed, tumor. Assays include (i) phenotypic analysis to determine the lineage of T cells in the cell product and in the blood (and tumor, if resection was performed) after infusion, (ii) quantification of the senescence and activation state of immune subsets from PBMCs, (iii) analysis of gene expression or epigenetic profiles to reflect the phenotype and functional state of the cells, and / or (iv) direct functional assessment of the cells. - Persistence of the infused genetically engineered cells and its correlation with therapeutic efficacy. Persistence can be determined by data on the number of copies of genetically modified DNA per μg of DNA and / or the number of transduced cells or total lymphocytes per μL. Well-established methodologies include (i) quantification of ADP-A2M4CD8 cells by quantitative PCR of the transgene from DNA extracted from frozen PBMCs and (ii) quantification and phenotypic analysis of ADP-A2M4CD8 cells by flow cytometry, DNA analysis, and RNA analysis from frozen PBMCs.

[0172] Figures 3 and 4 summarize the above process.

[0173] Max 10×10 9 Doses of ADP-A2M4CD8 were administered and shown to be well tolerated. New data indicate that when first-line treatment of newly metastatic or unresectable locally advanced bladder cancer includes administration of ADP-A2M4CD8, treatment outcomes may be improved. For example, outcomes may be improved compared to "standard of care" first-line treatment of newly metastatic or unresectable locally advanced bladder cancer.

[0174] Example 3. Preliminary clinical outcomes of next-generation autologous T cell receptor T cell therapy ADP-A2M4CD8 in patients with advanced urothelial cancer. Autologous T cells transduced with an autoinactivating lentiviral vector expressing a MAGE-A4-specific TCR and CD8α coreceptor were obtained by leukapheresis and reinfused into patients as ADP-A2M4CD8 after leukapheresis chemotherapy.

[0175] Seven patients with MAGE-A4-positive urothelial carcinoma were treated with ADP-A2M4CD8. Their baseline characteristics are shown in the table below:

[0176] [Table 1]

[0177] The overall response rate (ORR) according to investigator-reviewed Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 was 57.1% (one complete response, three partial responses; Figure 5 ).

[0178] The disease control rate was 100% (one complete response, three partial responses, and three stable disease).

[0179] The median duration of response was 19.14 weeks (range: 11.00-19.14 weeks). Two responses are ongoing. Data shown in Figure 5 represent the change from baseline SLD to disease progression or prior to surgical resection.

[0180] Sequence Listing SEQ ID NO: 1-MAGE-A4 230-239 GVYDGREHTV SEQ ID NO:2 - MAGE-A4 TCR alpha chain (CDRs are bold and underlined) VSPFSN , LTFSEN , CVVSGGTDSWGKLQF , signal sequence is italicized and underlined MKKHLTTFLVILWLYFYRGNG ) MKKHLTTFLVILWLYFYRGNG KNQVEQSPQSLIILEGKNCTLQCNYT VSPFSN LRWYKQDTGRGPVSLTI LTFSEN TKSNGRYTATLDADTKQSSLHITASQLSDSASYI CVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKR SEQ ID NO: 3 - MAGE-A4 TCR beta chain (CDRs are bold and underlined) KGHDR , SFDVKD , CATSGQGAYEEQFF , signal sequence is italicized and underlined MASLLFFCGAFYLLGTGSMDA ) MASLLFFCGAFYLLGTGSMDA DVTQTPRNRITKTGKRIMLECSQT KGHDR MYWYRQDPGLGLRLIYY SFDVKD INKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYF CATSGQGAYEEQFF GPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO:4 - MAGE-A4 TCR alpha chain CDR1 VSPFSN SEQ ID NO:5 - MAGE-A4 TCR alpha chain CDR2 LTFSEN SEQ ID NO:6 - MAGE-A4 TCR alpha chain CDR3 CVVSGGTDSWGKLQF SEQ ID NO:7-MAGE-A4 TCR beta chain CDR1 KGHDR SEQ ID NO:8 - MAGE-A4 TCR beta chain CDR2 SFDVKD SEQ ID NO: 9 - MAGE-A4 TCR beta chain CDR3 CATSGQGAYEEQFF SEQ ID NO: 10 - CD8 alpha chain (CDR-like loops are bold and underlined) VLLSNPTSG , YLSQNKPK , LSNSIM , signal sequence is italicized and underlined MALPVTALLLPLALLLHAARP ) MALPVTALLLPLALLLHAARP SQFRVSPLDRTWNLGETVELKCQ VLLSNPTSG CSWLFQPRGAAASPTFLL YLSQNKPK AAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSA LSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO:11 - CD8 alpha chain CDR1 VLLSNPTSG SEQ ID NO: 12 - CD8 alpha chain CDR2 YLSQNKPK SEQ ID NO: 13 - CD8 alpha chain CDR3 LSNSIM SEQ ID NO: 14 - PD1 - human programmed cell death protein (human) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCFSNTS ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS LVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL SEQ ID NO: 15 - PD1L1 - human programmed cell death 1 ligand 1 (human) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEME DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTT TTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTH LVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET SEQ ID NO: 16 - PD1L2 - human programmed cell death 1 ligand 2 (human) MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQ KVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL RLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATV IALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI SEQ ID NO: 17 - Nivolumab heavy chain sequence QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYY ADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK SEQ ID NO: 18 - Nivolumab light chain sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 19 - Pembrolizumab heavy chain sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNF NEKFKNRVTLTTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:20 - Pembrolizumab light chain sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 21 - Cemiplimab heavy chain sequence EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYF ADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 22 - Cemiplimab light chain sequence DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPS RFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFFGPGTVVDFRRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23 - Durvalumab heavy chain sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYY VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPPAFEG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:24 - Durvalumab light chain sequence EIVLTQSPGTLSLSPGERATLSCRASQRVSSYLAWYQQKPGQAPRLLIYDASSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 25 - Atezolizumab heavy chain sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 26 - Atezolizumab light chain sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27 - Avelumab heavy chain sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFY ADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 28 - Avelumab light chain sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVT LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 29 - MDX1105 heavy chain sequence QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT SEQ ID NO: 30 - MDX1105 light chain sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:31 - Dostallimab heavy chain sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMDYWGQGT TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 32 - Dostallimab light chain sequence DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A method of treating bladder cancer in an individual, comprising administering to said individual a population of modified T cells comprising a heterologous T cell receptor (TCR) capable of binding to a peptide antigen of MAGE-A4.

2. 10. The method of claim 1, further comprising administering a checkpoint inhibitor to the individual, optionally wherein the population of modified T cells and the checkpoint inhibitor are administered in the same selection therapy.

3. (a) administration of the checkpoint inhibitor is initiated prior to administration of the population of modified T cells and continued after administration of the population of modified T cells; or 3. The method of claim 2, wherein (b) administration of the checkpoint inhibitor is initiated simultaneously with or after administration of the population of modified T cells and is continued after administration of the population of modified T cells.

4. 4. The method of claim 2 or 3, wherein the checkpoint inhibitor comprises a programmed death-1 (PD-1) axis binding antagonist, optionally pembrolizumab.

5. administering an additional anti-cancer therapy to the individual, and optionally (a) the population of modified T cells and the additional anti-cancer therapy are administered in the same treatment regimen; and / or (b) The method of any one of the preceding claims, wherein the additional anti-cancer therapy is chemotherapy.

6. (a) administration of the additional anti-cancer therapy is initiated prior to administration of the population of modified T cells and continued after administration of the population of modified T cells; or 6. The method of claim 5, wherein (b) administration of the additional anti-cancer therapy is initiated simultaneously with or after administration of the population of modified T cells and is continued after administration of the population of modified T cells.

7. 10. The method of any one of the preceding claims, wherein the bladder cancer is recurrent bladder cancer.

8. the bladder cancer has recurred after curative-intent treatment for locally advanced bladder cancer, and optionally (a) the curative treatment comprises surgical resection and / or radiation therapy; and / or (b) The method of claim 7, wherein the curative treatment comprises systemic therapy.

9. (a) administering to the individual a checkpoint inhibitor, wherein administration of the checkpoint inhibitor is initiated simultaneously with or after administration of the population of modified T cells and continued after administration of the population of modified T cells; and / or 9. The method of claim 7 or 8, comprising (b) administering to the individual an additional anti-cancer therapy, wherein administration of the additional anti-cancer therapy is initiated simultaneously with or after administration of the population of modified T cells and continues after administration of the population of modified T cells, and optionally wherein the additional anti-cancer therapy is chemotherapy.

10. 7. The method of any one of claims 1-6, wherein the individual has not previously been treated for bladder cancer, and optionally, the bladder cancer is newly metastatic or unresectable locally advanced bladder cancer.

11. (a) administering to the individual a checkpoint inhibitor, wherein administration of the checkpoint inhibitor is initiated prior to or concurrently with administration of the population of modified T cells and continued after administration of the population of modified T cells; or 11. The method of claim 10, comprising (b) administering to the individual an additional anti-cancer therapy, wherein administration of the additional anti-cancer therapy is initiated prior to or concurrently with administration of the population of modified T cells and continued after administration of the population of modified T cells, and optionally wherein the additional anti-cancer therapy is chemotherapy.

12. (a) the population of modified T cells is administered as soon as possible after diagnosis of the bladder cancer; and / or (b) the method of any one of the preceding claims, wherein the population of modified T cells is administered as a single dose.

13. (a) the xenogeneic TCR binds to GVYDGREHTV (SEQ ID NO: 1) complexed with an HLA molecule; (b) the heterologous TCR comprises an alpha chain amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 and a beta chain amino acid sequence having at least 80% sequence identity to SEQ ID NO:3; and / or (c) the modified T cell further comprises a heterologous CD8 co-receptor, optionally wherein the heterologous CD8 co-receptor is CD8α.

14. the modified T cells are autologous with respect to the individual, and optionally, the method comprises: (a) obtaining peripheral blood mononuclear cells (PBMCs) from said individual; (b) selecting T cells from the PBMCs; (c) modifying the selected T cells to express said heterologous TCR and optionally a heterologous CD8 co-receptor; generating the population by Further optionally, the bladder cancer is recurrent bladder cancer and one or more of steps (a) to (c) are performed before recurrence.

15. 10. A population of modified T cells comprising a heterologous T cell receptor capable of binding to a peptide antigen of MAGE-A4, for use in a method according to any one of the preceding claims.

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