Methods of treating epstein barr virus associated lymphoproliferative disorders by t cell therapy

Abstract

The present invention relates to methods of treating Epstein-Barr virus-associated lymphoproliferative disorders by T cell therapy, specifically disclosed are methods of treating EBV-LPD (Epstein-Barr virus-associated lymphoproliferative disorder) in a human patient who has undergone a combination chemotherapy treatment for EBV-LPD and / or a radiation therapy treatment for EBV-LPD unsuccessfully. The methods comprise administering to the human patient an allogeneic T cell population comprising EBV-specific T cells.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on May 11, 2016, with application number 201680039509.4 and invention title "Method for treating Ebola virus-associated lymphoid proliferative disease by T-cell therapy".

[0002] Cross-references to related applications

[0003] This application claims the benefit of provisional application number 62 / 160,549, filed on May 12, 2015, which is incorporated herein by reference in its entirety.

[0004] Government Rights Statement

[0005] This invention was developed with government support, granted RO1 CA55349 by the National Institutes of Health (NIH). The government holds certain rights in this invention. Technical Field

[0006] This article discloses a method for treating human patients with EBV-LPD (Ebolavirus-associated lymphoproliferative disorder) who have experienced unsuccessful combination chemotherapy and / or radiotherapy for EBV-LPD, the method comprising administering an allogeneic T cell population containing EBV-specific T cells to the human patient. Background Technology

[0007] Epstein-Barr virus-associated lymphoproliferative disorders (EBV-LPD) are a major cause of morbidity and death in solid organ transplant recipients, hematopoietic stem cell transplant recipients, and other immunocompromised patients. Various therapies have been developed for treating EBV-LPD, such as chemotherapy, combination chemotherapy, radiotherapy, rituximab (an anti-CD20 monoclonal antibody) therapy, and cellular immunotherapy (see, for example, Elstrom et al., 2006, Am J Transplant 6:569-576; Haque et al., 2001, Transplantation 72:1399-1402; Haque et al., 2002, Lancet 360:435-442; Gandhi et al., 2007, American Journal of Transplantation 7:1293-1299; and Doubrovina, E. et al., Blood, 2012. 119: 2644-2656). When first-line therapy fails, subsequent lines of therapy are often attempted. For example, for many SOT receptors, especially those in patients with low-grade disease, first-line treatment involves reducing the dose of immunosuppressive drugs given to the patient. Several authors have reported the efficacy of rituximab monotherapy in patients who do not respond to dose reductions of immunosuppressants (see, for example, Webber et al., 2004, Blood 104: Abstract 746; and Messihel et al., 2006, Leuk Lymphoma 47:2584-2589). Opinions differ on the worthiness of single-agent rituximab retreatment if patients relapse after responding to or do not respond to rituximab, and many centers continue combination chemotherapy. The Children's Oncology Group recently completed a trial of low-dose cyclophosphamide, steroids, and rituximab, achieving a 2-year event-free survival (EFS) of 71% and an overall survival (OS) of 83% (Gross et al., 2012, Am J Transplant 12:3069-3075). In adult patients, treatment is more varied, including R-CHOP (a treatment regimen using cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) or DA-EPOCH (dose-adjusted EPOCH, a treatment regimen using etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin).In patients with CNS (central nervous system) involvement due to EBV-LPD, regimens include intrathecal rituximab alone for CNS disease alone or for systemic and CNS disease combination, radiation, or high-dose methotrexate.

[0008] EBV-LPD resistant to prior therapies (e.g., combination chemotherapy, radiotherapy, or rituximab) that have demonstrated clinical efficacy is more difficult to treat. The greater the clinically proven efficacy of prior therapies and the more the patient has experienced treatment failures, the greater the anticipated difficulty in achieving successful treatment with subsequent lines of therapy. EBV-LPD resistant to prior therapies is generally more aggressive. This is especially true when the prior therapy is chemotherapy or radiotherapy, which often produces or selects mutated tumor cells, leading to much larger and more aggressive disease. First-line therapy is typically chosen based on a combination of desired safety and efficacy, while subsequent lines of therapy are often considered less desirable in terms of their safety and / or efficacy profile. Therefore, there is a need to treat EBV-LPD in patients with a history of unsuccessful combination chemotherapy and / or radiotherapy with the desired safety and efficacy profile.

[0009] The citation of references in this document should not be construed as an admission that such references are prior art to this disclosure. Summary of the Invention

[0010] This invention relates to a method for treating human patients with EBV-LPD (Ebola virus-associated lymphoproliferative disorder) who have experienced unsuccessful combination chemotherapy and / or radiotherapy for EBV-LPD.

[0011] On one hand, this document provides a method for treating EBV-LPD in human patients, the method comprising administering an allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the human patient has experienced unsuccessful combination chemotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by human leukocyte antigen (HLA) alleles shared with cells of EBV-LPD. (The allogeneic T cell population is human). In some embodiments, EBV-LPD is resistant to combination chemotherapy. In some embodiments, the human patient withdraws from combination chemotherapy due to poor tolerance. In one specific embodiment, the combination chemotherapy that the human patient has experienced failure includes a therapy with cyclophosphamide and prednisone. In yet another specific embodiment, the combination chemotherapy that the human patient has experienced failure includes a low-dose cyclophosphamide and prednisone regimen. In another specific embodiment, the combination chemotherapy that the human patient has experienced failure includes a therapy with cyclophosphamide and methylprednisolone. In yet another specific embodiment, the combination chemotherapy that the human patient has experienced failure includes a low-dose cyclophosphamide and methylprednisolone regimen.

[0012] In specific implementations, human patients experience unsuccessful treatment with multiple different combination chemotherapy therapies for EBV-LPD. In one specific implementation, EBV-LPD develops resistance to multiple different combination chemotherapy therapies for EBV-LPD. In another specific implementation, human patients discontinue multiple different combination chemotherapy therapies due to poor tolerance. In one specific implementation, at least one of the failed multiple different combination chemotherapy therapies for human patients includes a therapy with cyclophosphamide and prednisone. In yet another specific implementation, at least one of the failed multiple different combination chemotherapy therapies for human patients includes a low-dose cyclophosphamide and prednisone regimen. In yet another specific implementation, at least one of the failed multiple different combination chemotherapy therapies for human patients includes a therapy with cyclophosphamide and methylprednisolone. In still another specific implementation, at least one of the failed multiple different combination chemotherapy therapies for human patients includes a low-dose cyclophosphamide and methylprednisolone regimen.

[0013] In specific implementations, human patients who have not responded to combination chemotherapy (or multiple different combination chemotherapy therapies) for EBV-LPD also do not respond to radiotherapy for EBV-LPD. In one specific implementation, EBV-LPD is resistant to radiotherapy for EBV-LPD. In another specific implementation, human patients withdraw from radiotherapy due to poor tolerance to radiotherapy.

[0014] On the other hand, this document provides a method for treating EBV-LPD in human patients, the method comprising administering an allogeneic T cell population containing EBV-specific T cells to the human patient; wherein the human patient has undergone unsuccessful radiotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by HLA alleles shared with cells of EBV-LPD. In some embodiments, EBV-LPD is resistant to radiotherapy for EBV-LPD. In some embodiments, the human patient withdraws from radiotherapy due to poor tolerance to radiotherapy.

[0015] In different implementations, EBV-LPD is a B-cell lineage cellular disease, in addition to failure to undergo the aforementioned combination chemotherapy (or multiple different combination chemotherapy therapies) and / or radiotherapy, and the human patient has also experienced unsuccessful treatment with anti-CD20 monoclonal antibodies. In some implementations, EBV-LPD is resistant to anti-CD20 monoclonal antibody treatment. In some implementations, the human patient discontinues anti-CD20 monoclonal antibody therapy due to poor tolerance. In one specific implementation, the anti-CD20 monoclonal antibody is rituximab.

[0016] In a specific implementation scheme, apart from being limited by HLA alleles shared with EBV-LPD, the allogeneic T cell population containing EBV-specific T cells shares at least two of the eight HLA alleles with EBV-LPD cells. In one specific implementation scheme, the eight HLA alleles are two HLA alleles, two HLA-B alleles, two HLA-C alleles, and two HLA-DR alleles.

[0017] In a specific implementation, the method for treating EBV-LPD described herein further includes a step of identifying at least one HLA allele of EBV-LPD cells by high-resolution typing prior to the administration step.

[0018] In different implementations, the method of treating EBV-LPD further includes a step of generating an allogeneic T cell population in vitro prior to the administration step.

[0019] In a specific implementation, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells to one or more EBV antigens.

[0020] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with EBV-transformed B cells. In one specific embodiment, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with EBV strain B95.8-transformed B cells.

[0021] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with dendritic cells, cytokine-activated monocytes, or peripheral blood mononuclear cells. In a specific embodiment, the step of sensitizing the allogeneic T cells with dendritic cells, cytokine-activated monocytes, or peripheral blood mononuclear cells includes loading the dendritic cells, cytokine-activated monocytes, or peripheral blood mononuclear cells with at least one immunogenic peptide derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing the allogeneic T cells with dendritic cells, cytokine-activated monocytes, or peripheral blood mononuclear cells includes loading the dendritic cells, cytokine-activated monocytes, or peripheral blood mononuclear cells with an overlapping peptide library derived from one or more EBV antigens.

[0022] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells using artificial antigen-presenting cells (AAPCs). In a specific embodiment, the step of sensitizing allogeneic T cells using AAPCs includes loading the AAPCs with at least one immunogenic peptide derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing allogeneic T cells using AAPCs includes loading the AAPCs with an overlapping peptide library derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing allogeneic T cells using AAPCs includes engineering the AAPCs to express at least one immunogenic EBV peptide or protein.

[0023] In a specific implementation plan, the step of generating an allogeneic T cell population in vitro further includes cryopreserving the allogeneic T cells after sensitization.

[0024] In a specific implementation, the method for treating EBV-LPD described herein further includes, prior to the administration step, thawing cryopreserved EBV-antigen-sensitized allogeneic T cells and expanding the allogeneic T cells in vitro to generate an allogeneic T cell population.

[0025] In some embodiments, the method for treating EBV-LPD described herein further includes a step of thawing a cryopreserved allogeneic T cell population prior to the administration step.

[0026] In different embodiments, the allogeneic T cell population is derived from a T cell line. In some embodiments, the method for treating EBV-LPD described herein further includes a step of selecting a T cell line from a variety of cryopreserved T cell line banks prior to the administration step. In some embodiments, the method for treating EBV-LPD described herein further includes a step of thawing the cryopreserved T cell line prior to the administration step. In a specific embodiment, the method for treating EBV-LPD described herein further includes a step of in vitro expansion of the T cell line prior to the administration step.

[0027] In a specific implementation scheme, the EBV antigen recognized by the EBV-specific T cells administered according to the method described herein is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, or LMP2.

[0028] In some embodiments, administration is via infusion of an allogeneic T-cell population. In some embodiments, the infusion is an intravenous bolus. In some embodiments, administration includes administering at least about 1 x 10⁻⁶ cells. 5 Administer approximately 1 x 10n allogeneic T cell populations per kg per dose per week to a human patient. In some embodiments, administration includes approximately 1 x 10n T cells per kg per dose per week. 6- Approximately 2 x 10 6 One allogeneic T cell population per kg of T cells per week is administered to a human patient. In one specific implementation, administration includes administering approximately 1 x 102 T cells per kg per dose per week. 6 Administer approximately 2 x 10 allogeneic T cell populations per kg per dose per week to a human patient. In another specific implementation, administration includes administering approximately 2 x 10 6 One allogeneic T cell population per kg of T cells per week was administered to a human patient.

[0029] In some embodiments, the method of treating EBV-LPD described herein includes administering at least two doses of allogeneic T cell populations to a human patient. In specific embodiments, the method of treating EBV-LPD described herein includes administering 2, 3, 4, 5, or 6 doses of allogeneic T cell populations to a human patient.

[0030] In some embodiments, the method of treating EBV-LPD described herein includes a first cycle of administering one dose of allogeneic T-cell population once weekly for three consecutive weeks, followed by an interval during which no dose of allogeneic T-cell population is administered, followed by a second cycle of administering one dose of allogeneic T-cell population once weekly for three consecutive weeks. In specific embodiments, the method of treating EBV-LPD described herein includes administering one dose of allogeneic T-cell population once weekly for two, three, four, five, or six cycles, each cycle being separated by an interval during which no dose of allogeneic T-cell population is administered. In one specific embodiment, the interval is approximately three weeks.

[0031] In some embodiments, the method of treating EBV-LPD further includes administering a second allogeneic T cell population comprising EBV-specific T cells to the human patient following administration of an allogeneic T cell population; wherein the second allogeneic T cell population is restricted by different HLA alleles shared with the cells of EBV-LPD. In one specific embodiment, the method of treating EBV-LPD comprises administering one dose of the allogeneic T cell population weekly for three consecutive weeks in a first cycle, followed by an interval during which no dose of the allogeneic T cell population is administered, followed by a second cycle of one dose of the second allogeneic T cell population weekly for three consecutive weeks. In yet another specific embodiment, the interval is approximately three weeks.

[0032] In some implementations, human patients do not respond, have an incomplete response, or a suboptimal response after administration of an allogeneic T cell population and before administration of a second allogeneic T cell population.

[0033] Human patients can be anyone who has EBV-LPD and has experienced unsuccessful combination chemotherapy (and in some embodiments, unsuccessful therapy with anti-CD20 monoclonal antibodies) and / or unsuccessful radiation therapy (and in some embodiments, unsuccessful therapy with anti-CD20 monoclonal antibodies).

[0034] In one specific implementation, EBV-LPD is EBV-positive lymphoma. In another specific implementation, EBV-LPD treated according to the methods described herein is present in the central nervous system of a human patient. In yet another specific implementation, EBV-LPD treated according to the methods described herein is present in the brain of a human patient.

[0035] In some implementations, the human patient was a recipient of a solid organ transplant from a transplant donor. In some implementations, the human patient was a recipient of a multi-organ transplant (e.g., heart-lung transplant or kidney-pancreas transplant). Solid organ transplants may include, but are not limited to, kidney transplants, liver transplants, heart transplants, intestinal transplants, pancreas transplants, lung transplants, or combinations thereof. In one specific implementation, the solid organ transplant is a kidney transplant. In another specific implementation, the solid organ transplant is a liver transplant. In some implementations, the human patient was a recipient of a hematopoietic stem cell transplant (HSCT) from a transplant donor. HSCT can be a bone marrow transplant, a peripheral blood stem cell transplant, or a cord blood transplant. In a specific implementation, the allogeneic T cell population is derived from a donor other than the transplant donor. Detailed Implementation

[0036] This invention relates to a method for treating EBV-LPD (Ebola virus-associated lymphoid disease) in human patients who have experienced unsuccessful treatment with combination chemotherapy and / or radiotherapy for EBV-LPD. The invention provides an effective T-cell therapy for treating EBV-LPD resistant to combination chemotherapy or radiotherapy, and is therefore intended for use as a low-toxicity second-line therapy.

[0037] On one hand, this document provides a method for treating EBV-LPD in human patients, the method comprising administering an allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the human patient has unsuccessfully undergone combination chemotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by human leukocyte antigen (HLA) alleles common to cells with EBV-LPD. In some embodiments, EBV-LPD is resistant to combination chemotherapy. In some embodiments, the human patient withdraws from combination chemotherapy due to poor tolerance. In specific embodiments, the human patient has unsuccessfully undergone multiple different combination chemotherapies for EBV-LPD. In one specific embodiment, EBV-LPD is resistant to multiple different combination chemotherapies for EBV-LPD. In another specific embodiment, the human patient withdraws from multiple different combination chemotherapies due to poor tolerance. In a specific embodiment, the human patient has unsuccessfully undergone radiotherapy for EBV-LPD. In one specific embodiment, EBV-LPD is resistant to radiotherapy for EBV-LPD. In another specific implementation, human patients withdraw from radiotherapy due to poor tolerance to the therapy.

[0038] On the other hand, this document provides a method for treating EBV-LPD in human patients, the method comprising administering an allogeneic T cell population containing EBV-specific T cells to the human patient; wherein the human patient has undergone unsuccessful radiotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by HLA alleles shared with cells of EBV-LPD. In some embodiments, EBV-LPD is resistant to radiotherapy for EBV-LPD. In some embodiments, the human patient withdraws from radiotherapy due to poor tolerance to radiotherapy.

[0039] In different implementations, EBV-LPD is a B-cell lineage cellular disease in addition to failure to undergo the aforementioned combination chemotherapy (or multiple different combination chemotherapy therapies) and / or radiotherapy, and the human patient has also experienced unsuccessful treatment with anti-CD20 monoclonal antibodies (e.g., rituximab). In some implementations, EBV-LPD is resistant to treatment with anti-CD20 monoclonal antibodies. In some implementations, human patients discontinue anti-CD20 monoclonal antibody therapy due to poor tolerance.

[0040] A human patient is considered to have experienced unsuccessful treatment with EBV-LPD (e.g., combination chemotherapy, radiotherapy, and / or anti-CD20 monoclonal antibody therapy) if they are resistant to a particular therapy and / or if they withdraw from therapy due to poor tolerance to a particular therapy (e.g., due to the toxicity of the therapy given to the patient's age or condition). EBV-LPD is considered resistant to a therapy (e.g., combination chemotherapy, radiotherapy, or anti-CD20 monoclonal antibody therapy) if there is no response to EBV-LPD, or an incomplete response (less than a complete remission), or progression, or relapse after treatment. A complete remission is the complete disappearance of all clinical and radiological signs of the disease (optionally confirmed by a biopsy of the affected tissue) for at least 3 weeks after completion of treatment.

[0041] 1.1. Combination chemotherapy, radiotherapy, and anti-CD20 antibodies

[0042] Combination chemotherapy involves the therapeutic application of treating cancer during the same treatment period with two or more different chemotherapeutic agents. Chemotherapy agents are chemical drugs that act against cancer cells; they are generally synthetic small-molecule organic compounds, distinct from other types of anticancer agents (such as biopolymers and cells). Therefore, chemotherapeutic agents are not nucleic acids, proteins (such as antibodies), or immune cells (such as T cells). Combination chemotherapy is often attempted to minimize resistance to the therapy in cancers such as EBV-LPD. This is because cancer cells can mutate and become resistant to a single chemotherapeutic agent, but by using different chemotherapeutic agents, it may be more difficult for the cancer to mutate to develop resistance to the combination. Therefore, EBV-LPD resistant to combination chemotherapy is generally considered more difficult to treat than EBV-LPD resistant to a single agent.

[0043] This invention provides treatment for human patients suffering from EBV-LPD who have experienced unsuccessful combination chemotherapy for EBV-LPD. The unsuccessful combination chemotherapy for human patients can be any combination chemotherapy known in the art for the treatment of LPD (lymphoproliferative disorder). Exemplary combination chemotherapy therapies include, but are not limited to (the combination is the chemotherapy agent in parentheses): 7+3 (7 days of cytarabine plus 3 days of anthracycline antibiotics, or daunorubicin or idarubicin), ABVD (doxorubicin, bleomycin, vincristine, dacarbazine), BACOD (bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone), BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone), dose-escalating BEACOPP, CBV (cyclophosphamide, carmustine, etoposide), COP (cyclophosphamide, vincristine and prednisone or prednisolone), CHOEP (cyclophosphamide, doxorubicin, etoposide, vincristine, prednisone), CEOP (cyclophosphamide, etoposide, vincristine, prednisone), CEPP (cyclophosphamide, etoposide, procarbazine, prednisone), ChlVPP (Chloramine mustard, vincristine, procarbazine, prednisone, etoposide, vinblastine, doxorubicin), CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), DCEP (dexamethasone, cyclophosphamide, etoposide, platinum), DHAP (dexamethasone, cytarabine, platinum), DICE (dexamethasone, ifosfamide, cisplatin, etoposide), DT-PACE (dexamethasone, thalidomide, platinum, doxorubicin, cyclophosphamide, etoposide), EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin), DA-EPOCH (dose-adjusted EPOCH), ESHAP (etoposide, methylprednisolone, cytarabine, cisplatin), FCM (fludarabine, cyclophosphamide, mitoxantrone), FM (fludarabine, mitoxantrone), FLAG (Fludarabine, Cytarabine, G-CSF), FLAG-IDA (Fludarabine, Cytarabine, Idarubicin, G-CSF), FLAG-MITO (Mitoxantrone, Fludarabine, Cytarabine, G-CSF), FLAMSA (Fludarabine, Cytarabine, Acridine), FLAMSA-BU (Fludarabine, Cytarabine, Acridine, Busulfan), FLAMSA-MEL (Fludarabine, Cytarabine, Acridine, Melphalan), GVD (Gemcitabine, Vinorelbine, PEGylated Liposome Doxorubicin), GEMOX (Gemcitabine, Oxaliplatin), IAC (Idarubicin x 3 days plus Cytarabine x 7 days), ICE (Ifosfamide, Carboplatin, Etoposide), IVAC (Etoposide, Cytarabine, Ifosfamide), m-BACOD(Methotrexate, Bleomycin, Doxorubicin, Cyclophosphamide, Vincristine, Dexamethasone), MACOP-B (Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide, Vincristine, Prednisone, Bleomycin), MINE (Mesna, Ifosfamide, Nootropine, Etoposide), MOPP (Nitrogen Mustard, Vincristine, Procarbazine, Prednisone), MVP (Mitomycin, Vincristine, Cisplatin), PACE (Platinum, Doxorubicin, Cyclophosphamide, Etoposide), PEB (Cisplatin, Etoposide, Bleomycin), POMP (6-Mercaptopurine, Vincristine, Methotrexate, Prednisone), ProMACE-MOPP (Methotrexate, Doxorubicin, Cyclophosphamide, Etoposide, Nitrogen Mustard, Vincristine, Procarbazine, Prednisone), ProMACE-CytaBOM (Prednisone, Doxorubicin, Cyclophosphamide, Etoposide, Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin), RVD (Lenalidomide, Bortezomib, Dexamethasone), Stanford V (Doxorubicin, Nitrogen Mustard, Bleomycin, Vincristine, Vincristine, Etoposide, Prednisone), Thal / Dex (Thalidomide, Dexamethasone), VAD (Vincristine, Doxorubicin, Dexamethasone), VAMP (Vincristine, Methotrexate, 6-Mercaptopurine and Prednisone, or Vincristine, Doxorubicin, Methotrexate and Prednisone, or Vincristine, Doxorubicin and Methylprednisolone), VAPEC-B (Vincristine, Doxorubicin, Prednisone, Etoposide, Cyclophosphamide, Bleomycin), VD-PACE (bortezomib, dexamethasone, platinum, doxorubicin, cyclophosphamide, etoposide) and VTD-PACE (bortezomib, thalidomide, dexamethasone, platinum, doxorubicin, cyclophosphamide, etoposide).

[0044] In one specific implementation, the human patient who experienced unsuccessful combination chemotherapy was treated with cyclophosphamide and prednisone. In another specific implementation, the human patient who experienced unsuccessful combination chemotherapy was treated with a low-dose cyclophosphamide and prednisone regimen. The low-dose cyclophosphamide and prednisone regimen involves administering less than approximately 900 mg / m². 2 The regimen involves administering less than 8 doses of intravenous cyclophosphamide and less than 2 mg / kg / dose of oral prednisone twice daily. In one specific implementation, the combination chemotherapy is described in Gross et al., 2012, Am J Transplant 12:3069-3075, as a low-dose cyclophosphamide and prednisone regimen, as follows: a total of 6 cycles of therapy, administered every 3 weeks; 600 mg / m² 2 Intravenous cyclophosphamide was administered on the first day of each cycle, and oral prednisone at 1 mg / kg was administered twice daily from day 1 to day 5 of each cycle.

[0045] In one specific implementation, the combination chemotherapy that the human patient experienced was unsuccessful was a combination of cyclophosphamide and methylprednisolone. In one specific implementation, the combination chemotherapy that the human patient experienced unsuccessful was a low-dose cyclophosphamide and methylprednisolone regimen. In one specific implementation, the combination chemotherapy was the low-dose cyclophosphamide and methylprednisolone regimen described in Gross et al., 2012, Am J Transplant 12:3069-3075, as follows: administration of a total of 6 cycles of therapy, with each cycle administered every 3 weeks; 600 mg / m². 2 Intravenous cyclophosphamide was administered on the first day of each cycle, and intravenous methylprednisolone 0.8 mg / kg was administered every 12 hours from day 1 to day 5 of each cycle.

[0046] In one specific implementation, the unsuccessful combination chemotherapy in human patients was with cyclophosphamide, prednisone, and methylprednisolone. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with gemcitabine and vinorelbine. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with methotrexate and temozolomide. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with methotrexate, temozolomide, and cytarabine. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with prednisone and cyclophosphamide. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with vincristine and cyclophosphamide. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with doxorubicin, vincristine, prednisone, and methotrexate. In one specific implementation, the unsuccessful combination chemotherapy in human patients was with vincristine, lomustine, and cytarabine. In one specific implementation, the unsuccessful combination chemotherapy in human patients was COP. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was BEACOPP. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was CHOP. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was a combination of cyclophosphamide, doxorubicin, vincristine, prednisone, cytarabine, methotrexate, and dexamethasone. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was IVAC. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was ESHAP. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was a combination of melphalan and dexamethasone. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was ProMACE-CytaBOM. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was CHOP. In one specific implementation, the combination chemotherapy that was unsuccessful for human patients was DA-EPOCH.

[0047] In one specific implementation, unsuccessful combination chemotherapy in human patients includes any combination of the aforementioned chemotherapeutic agents or chemotherapy regimens. In another specific implementation, unsuccessful combination chemotherapy in human patients consists primarily of any combination of the aforementioned chemotherapeutic agents or chemotherapy regimens.

[0048] In one specific implementation, when a human patient has failed to respond to multiple different combination chemotherapy therapies for EBV-LPD, at least one of the multiple different combination chemotherapy therapies is any combination of the aforementioned chemotherapeutic agents or chemotherapy regimens. In one specific implementation, when a human patient has failed to respond to multiple different combination chemotherapy therapies for EBV-LPD, at least one of the multiple different combination chemotherapy therapies includes any combination of the aforementioned chemotherapeutic agents or chemotherapy regimens. In one specific implementation, when a human patient has failed to respond to multiple different combination chemotherapy therapies for EBV-LPD, at least one of the multiple different combination chemotherapy therapies is substantially composed of any combination of the aforementioned chemotherapeutic agents or chemotherapy regimens.

[0049] Radiation therapy uses high-energy radiation to kill cancer cells by destroying their DNA. According to the present invention, unsuccessful radiation therapy experienced by human patients can be any of the types known in the art for treating LPD. Exemplary radiation therapies include, but are not limited to: conventional external beam radiation therapy, stereotactic radiation therapy, intensity-modulated radiation therapy, volume-modulated arc therapy, particle therapy, Auger therapy, brachytherapy, and radioisotope therapy.

[0050] In various embodiments, EBV-LPD is also a B-cell lineage disease, in addition to the failure of any of the aforementioned combination chemotherapy and / or radiotherapy, and the human patient has also experienced unsuccessful treatment with anti-CD20 monoclonal antibodies (alone or in combination with other therapies targeting EBV-LPD). The anti-CD20 monoclonal antibody can be any monoclonal antibody known in the art. In specific embodiments, the anti-CD20 monoclonal antibody is a chimeric antibody or a humanized antibody. In specific embodiments, the anti-CD20 monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In some embodiments, the anti-CD20 monoclonal antibody is a monospecific antibody or a multispecific (e.g., bispecific) antibody. In specific embodiments, the anti-CD20 monoclonal antibody is conjugated to a cytotoxic agent; alternatively, the anti-CD20 monoclonal antibody may be unconjugated. Exemplary anti-CD20 monoclonal antibodies include, but are not limited to: rituximab, obinutuzumab, ocrelizumab, temozumab, tosimob, and veltuzumab. In one specific implementation, the anti-CD20 monoclonal antibody is rituximab. In one specific implementation, a human patient experienced unsuccessful treatment with R-CEOP (a regimen of cyclophosphamide, etoposide, vincristine, prednisone, and rituximab). In one specific implementation, a human patient experienced unsuccessful treatment with R-GEMOX (a regimen of gemcitabine, oxaliplatin, and rituximab). In one specific implementation, a human patient experienced unsuccessful treatment with R-COP (a regimen of cyclophosphamide, vincristine, prednisone / prednisolone, and rituximab). In one specific implementation, a human patient experienced unsuccessful treatment with R-CHOP (a regimen of cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab). In one specific implementation, a human patient experienced unsuccessful treatment with rituximab, cyclophosphamide, and prednisone. In one specific implementation, a human patient experienced unsuccessful treatment with rituximab, cyclophosphamide, and methylprednisolone. In one specific implementation, the treatment regimen described in Gross et al., 2012, Am J Transplant 12:3069-3075 was unsuccessful in human patients, as follows: a total of 6 cycles of therapy were administered, with each cycle administered every 3 weeks; 600 mg / m² 2 Intravenous cyclophosphamide was administered on the first day of each cycle for up to 6 cycles, and oral prednisone 1 mg / kg was administered twice daily on days 1-5 of each cycle (or intravenous methylprednisolone 0.8 mg / kg every 12 hours) for up to 6 cycles, with a dose of 375 mg / m 2 Intravenous rituximab was administered on days 1, 8, and 15 of each cycle for the first two cycles.

[0051] As mentioned above, it will be clear that when human patients experience unsuccessful treatment with both combination chemotherapy for EBV-LPD and anti-CD20 monoclonal antibody therapy for EBV-LPD, combination chemotherapy and anti-CD20 monoclonal antibody therapy can be combined in a single treatment regimen or in combination with various treatment regimens administered to human patients at different times.

[0052] 1.2. Allogeneic T cell populations restricted by HLA alleles shared with EBV-LPD

[0053] According to the present invention, an allogeneic T cell population comprising EBV-LPD-specific T cells is administered to a human patient. The allogeneic T cell population administered to the human patient is restricted by HLA alleles shared with EBV-LPD cells. In some embodiments, this HLA allele restriction is ensured by determining the HLA assignment of EBV-LPD cells and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) comprising EBV-specific T cells restricted by the HLA alleles of such cells. In other embodiments, when it is determined that the HLA assignment of EBV-LPD cells is impossible and the human patient has not been a recipient of transplantation, this HLA allele restriction is ensured by determining the HLA assignment of the human patient (e.g., by using non-LPD cells or tissue from the human patient) and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) comprising EBV-specific T cells restricted by the HLA alleles of the human patient. In other implementations, when the HLA localization of EBV-LPD cells is not determined and a human patient was previously a recipient of the transplant, this HLA allele restriction is ensured by determining the source of EBV-LPD (whether transplant donor or recipient (human patient)), determining the HLA localization of the source of EBV-LPD (transplant donor or human patient, as the case may be), and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) containing EBV-LPD-derived HLA alleles that are restricted by those alleles. In these implementations, when the source of EBV-LPD is not determined, this HLA allele restriction is ensured by determining the HLA localization of both the human patient and the transplant donor, and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) containing EBV-derived T cells that are restricted by HLA alleles shared by both the human patient and the transplant donor.

[0054] The origin of EBV-LPD can be determined by any method known in the art, such as by analyzing variable tandem repeats (VTRs), a method that distinguishes transplant recipients and donors using a unique DNA signature of small DNA sequences from different individuals; or, if the transplant recipients and donors are of different sexes, by determining the presence or absence of chromosome Y (through cytogenetics or FISH (fluorescence in situ hybridization)).

[0055] In some embodiments for determining HLA localization, at least four HLA loci (preferably HLA-A, HLA-B, HLA-C, and HLA-DR) are genotyped. In some embodiments, four HLA loci (preferably HLA-A, HLA-B, HLA-C, and HLA-DR) are genotyped. In some embodiments, six HLA loci are genotyped. In some embodiments, eight HLA loci are genotyped.

[0056] In specific implementations, in addition to being limited by HLA alleles shared with EBV-LPD, allogeneic T cell populations containing EBV-specific T cells share at least two of the eight HLA alleles with EBV-LPD cells (e.g., two HLA alleles, two HLA-B alleles, two HLA-C alleles, and two HLA-DR alleles). In some implementations, this sharing is ensured by determining the HLA localization of EBV-LPD cells and selecting allogeneic T cell populations (or T cell lines from which allogeneic T cell populations are derived) of EBV-specific T cells that share at least two of the eight HLA alleles with these cells. In other embodiments, when it is determined that the HLA localization of EBV-LPD cells is impossible and the human patient has never been a recipient of transplantation, this commonality is ensured by determining the HLA localization of the human patient (e.g., by using non-LPD cells or tissue from the human patient) and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) containing EBV-specific T cells that share at least two of the eight HLA alleles with the human patient. In other embodiments, when it is determined that the HLA localization of EBV-LPD cells is impossible and the human patient has previously been a recipient of transplantation, this commonality is ensured by determining the source of EBV-LPD (whether transplant donor or recipient (human patient)), determining the HLA localization of the source of EBV-LPD (transplant donor or human patient, as appropriate), and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) containing EBV-specific T cells that share at least two of the eight HLA alleles with the source of EBV-LPD. In such implementations, when it is not possible to determine the source of EBV-LPD, this is ensured by determining the HLA localization of both the human patient and the transplant donor, and selecting an allogeneic T cell population (or a T cell line from which an allogeneic T cell population is derived) containing at least two of the eight HLA alleles shared by both the human patient and the transplant donor.

[0057] HLA locus type (i.e., HLA typing) can be determined by any method known in the art. Non-limiting exemplary methods for determining HLA localization can be found in ASHI Laboratory Manual, Edition 4.2 (2003), American Society for Histocompatibility and Immunogenetics; ASHI Laboratory Manual, Supplements 1 (2006) and 2 (2007), American Society for Histocompatibility and Immunogenetics; Hurley, “DNA-based typing of HLA for transplantation.” Edited by Leffell et al., 1997, Handbook of Human Immunology, Boca Raton: CRC Press; Dunn, 2011, Int J Immunogenet 38:463-473; Erlich, 2012, Tissue Antigens, 80:1-11; Bontadini, 2012, Methods, 56:471-476; and Lange et al., 2014, BMC Genomics 15: 63.

[0058] In general, high-resolution typing is preferred for HLA typing. High-resolution typing can be performed by any method known in the art, such as those described in: ASHI Laboratory Manual, Edition 4.2 (2003), American Society for Histocompatibility and Immunogenetics; ASHI Laboratory Manual, Supplements 1 (2006) and 2 (2007), American Society for Histocompatibility and Immunogenetics; Flomenberg et al., Blood, 104:1923-1930; Kögler et al., 2005, Bone Marrow Transplant, 36:1033-1041; Lee et al., 2007, Blood 110:4576-4583; Erlich, 2012, Tissue Antigens, 80:1-11; Lank et al., 2012, BMC Genomics 13:378; or Gabriel et al., 2014, Tissue Antigens, 83:65-75. In a specific implementation, the method for treating EBV-LPD described herein further includes a step of identifying at least one HLA allele of EBV-LPD cells by high-resolution typing prior to the administration step.

[0059] The HLA alleles that restrict allogeneic T cell populations can be determined by any method known in the art, such as those described in: Trivedi et al., 2005, Blood 105:2793-2801; Barker et al., 2010, Blood 116:5045-5049; Hasan et al., 2009, J Immunol, 183:2837-2850; or Doubrovina et al., 2012, Blood 120:1633-1646.

[0060] The preferred method is to define HLA alleles shared by the allogeneic T cell population and EBV-LPD cells using high-resolution typing. The preferred method is to define HLA alleles shared between the allogeneic T cell population and EBV-LPD cells using high-resolution typing. The most preferred method is to define HLA alleles shared by the allogeneic T cell population and EBV-LPD cells, as well as HLA alleles shared between the allogeneic T cell population and EBV-LPD cells, using high-resolution typing.

[0061] 1.3. Obtain or generate an allogeneic T cell population containing EBV-specific T cells

[0062] The allogeneic T cell population containing EBV-specific T cells, to be administered to human patients, can be generated by any method known in the art, or can be generated by selecting from an existing cryopreserved T cell line (each T cell line containing EBV-specific T cells) bank (collection) generated by any method known in the art, thawing it, and preferably expanding it prior to administration. Preferably, the unique markers of each T cell line in the bank relate to information about the HLA alleles restricted to the corresponding T cell line, and optionally, information about the HLA localization of the corresponding T cell line. The allogeneic T cell population and T cell lines in the bank are preferably obtained or generated by the methods described below.

[0063] In different implementations, the method of treating EBV-LPD further includes a step of obtaining an allogeneic T cell population prior to the administration step.

[0064] In a specific implementation, the step of obtaining an allogeneic T cell population includes sorting EBV-positive T cells from a blood cell population using fluorescently activated cells. In one specific implementation, the blood cell population consists of peripheral blood mononuclear cells (PBMCs) isolated from a blood sample obtained from a human donor. Fluorescently activated cell sorting can be performed by any method known in the art, which generally includes staining the blood cell population with an antibody that recognizes at least one EBV antigen prior to the sorting step.

[0065] In a specific implementation, the step of obtaining an allogeneic T cell population includes generating the allogeneic T cell population in vitro. The allogeneic T cell population can be generated in vitro by any method known in the art. Non-limiting exemplary methods for generating an allogeneic T cell population can be found in Koehne et al., 2000, Blood 96:109-117; Koehne et al., 2002, Blood 99:1730-1740; O'Reilly et al., 2007, Immunol Res 38:237-250; Barker et al., 2010, Blood 116:5045-5049; O'Reilly et al., 2011, Best Practice & Research ClinicalHaematology 24:381-391; and Doubrovina et al., 2012, Blood 119:2644-2656.

[0066] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing (i.e., stimulating) allogeneic T cells to one or more EBV antigens to generate EBV-specific T cells. Allogeneic T cells for generating an allogeneic T cell population in vitro can be isolated from an allogeneic T cell donor by any method known in the art, such as those described in Koehne et al., 2002, Blood 99:1730-1740; O'Reilly et al., 2007, Immunol Res. 38:237-250; or Barker et al., 2010, Blood 116:5045-5049. In one specific embodiment, allogeneic T cells are enriched from peripheral blood lymphocytes isolated from PBMCs of an allogeneic T cell donor. In yet another specific embodiment, T cells are enriched from peripheral blood lymphocytes isolated from PBMCs of an allogeneic T cell donor by depleting adherent monocytes followed by natural killer cells. In various embodiments, allogeneic T cells are cryopreserved for storage. In one specific embodiment, allogeneic T cells are cryopreserved, thawed, and expanded in vitro prior to sensitization. In another specific embodiment, allogeneic T cells are cryopreserved, thawed, and then sensitized, but not expanded in vitro prior to sensitization, followed optionally by expansion. In a specific embodiment, allogeneic T cells are cryopreserved after sensitization (sensitization produces EBV-specific T cells). In one specific embodiment, allogeneic T cells are cryopreserved after sensitization, thawed, and expanded in vitro to produce an allogeneic T cell population containing EBV-specific T cells. In yet another specific embodiment, allogeneic T cells are cryopreserved after sensitization, thawed, but not expanded in vitro to produce an allogeneic T cell population containing EBV-specific T cells. In other different embodiments, allogeneic T cells are not cryopreserved. In one specific implementation, the allogeneic T cells are not cryopreserved, and they are expanded in vitro prior to sensitization. In another specific implementation, the allogeneic T cells are not cryopreserved, and they are not expanded in vitro prior to sensitization. In yet another specific implementation, the step of generating an allogeneic T cell population in vitro further includes cryopreserving the allogeneic T cells after sensitization.

[0067] In a specific implementation, the method for treating EBV-LPD described herein further includes, prior to the administration step, thawing cryopreserved EBV-antigen-sensitized allogeneic T cells and expanding the allogeneic T cells in vitro to generate an allogeneic T cell population.

[0068] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with EBV-transformed B cells (i.e., exposing the allogeneic T cells to EBV-transformed B cells). For example, B cells transformed with EBV strain B95.8 can be used for this purpose.

[0069] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with dendritic cells (preferably derived from an allogeneic T cell donor). In a specific embodiment, the step of sensitizing the allogeneic T cells with dendritic cells includes loading the dendritic cells with at least one immunogenic peptide derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing the allogeneic T cells with dendritic cells includes loading the dendritic cells with an overlapping peptide library derived from one or more EBV antigens.

[0070] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with cytokine-activated monocytes (preferably derived from an allogeneic T cell donor). In a specific embodiment, the step of sensitizing the allogeneic T cells with cytokine-activated monocytes includes loading the cytokine-activated monocytes with at least one immunogenic peptide derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing the allogeneic T cells with cytokine-activated monocytes includes loading the cytokine-activated monocytes with an overlapping peptide library derived from one or more EBV antigens.

[0071] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with peripheral blood mononuclear cells (preferably derived from an allogeneic T cell donor). In a specific embodiment, the step of sensitizing the allogeneic T cells with peripheral blood mononuclear cells includes loading the peripheral blood mononuclear cells with at least one immunogenic peptide derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing the allogeneic T cells with peripheral blood mononuclear cells includes loading the peripheral blood mononuclear cells with an overlapping peptide library derived from one or more EBV antigens.

[0072] In some embodiments, the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells using artificial antigen-presenting cells (AAPCs). In a specific embodiment, the step of sensitizing allogeneic T cells using AAPCs includes loading the AAPCs with at least one immunogenic peptide derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing allogeneic T cells using AAPCs includes loading the AAPCs with an overlapping peptide library derived from one or more EBV antigens. In a specific embodiment, the step of sensitizing allogeneic T cells using AAPCs includes engineering the AAPCs to express at least one immunogenic EBV peptide or protein.

[0073] In different embodiments, the peptide library is an overlapping peptide library of antigens spanning EBV. In different embodiments, the peptide library is an overlapping peptide library of more than one antigen spanning EBV. In one specific embodiment, the overlapping peptide library is an overlapping 15-peptide library.

[0074] In specific embodiments, the allogeneic T cell population is cryopreserved for storage prior to administration. In specific embodiments, the allogeneic T cell population is not cryopreserved for storage prior to administration. In some embodiments, the method for treating EBV-LPD described herein further includes a step of thawing the cryopreserved allogeneic T cell population prior to the administration step.

[0075] In different embodiments, the allogeneic T cell population is derived from a T cell line. In a specific embodiment, the T cell line is cryopreserved for storage prior to administration. In a specific embodiment, the T cell line is not cryopreserved for storage prior to administration. In some embodiments, the T cell line is expanded in vitro to obtain an allogeneic T cell population. In other embodiments, the T cell line is not expanded in vitro to obtain an allogeneic T cell population. The T cell line may be sensitized to one or more EBV antigens (to generate EBV-specific T cells, for example, through the sensitization step described above) before or after cryopreservation (if the T cell line is cryopreserved) or before or after in vitro expansion (if the T cell line is expanded in vitro). In some embodiments, the method for treating EBV-LPD described herein further includes the step of selecting a T cell line from a plurality of cryopreserved T cell line libraries (preferably each containing EBV-specific T cells) prior to the administration step. The unique identifiers of each T cell line in the preferred library are related to information about the HLA alleles restricted to the corresponding T cell line and, optionally, information about the HLA localization of the corresponding T cell line. In some embodiments, the method for treating EBV-LPD described herein further includes a step of thawing a cryopreserved T cell line prior to the administration step. In specific embodiments, the method for treating EBV-LPD described herein further includes a step of in vitro expansion of the T cell line (e.g., after thawing the cryopreserved T cell line) prior to the administration step. T cell lines and various cryopreserved T cell lines can be generated by any method known in the art, such as those described in Koehne et al., 2002, Blood 99:1730-1740; O'Reilly et al., 2007, Immunol Res. 38:237-250; Barker et al., 2010, Blood 116:5045-5049, or as described above regarding the generation of allogeneic T cell populations in vitro.

[0076] The allogeneic T cell population containing EBV-specific T cells given to human patients includes CD8+ T cells, and in one specific implementation, it also includes CD4+ T cells.

[0077] EBV-specific T cells administered according to the method described herein recognize at least one EBV antigen. In a specific implementation, the EBV-specific T cells administered according to the method described herein recognize the EBV antigen EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, or LMP2.

[0078] 1.4. Administration and Dosage

[0079] The route of administration and the dosage of the allogeneic T cell population to be given to the human patient can be determined based on the condition of the human patient and the physician's knowledge. Administration is generally intravenous.

[0080] In some embodiments, administration is via infusion of an allogeneic T-cell population. In some embodiments, the infusion is an intravenous bolus. In some embodiments, administration includes administering at least about 1 x 10⁻⁶ cells. 5 Administer approximately 1 x 10n allogeneic T cell populations per kg per dose per week to a human patient. In some embodiments, administration includes approximately 1 x 10n T cells per kg per dose per week. 6 - Approximately 2 x 10 6 One allogeneic T cell population per kg of T cells per week is administered to a human patient. In one specific implementation, administration includes administering approximately 1 x 102 T cells per kg per dose per week. 6 Administer approximately 2 x 10 allogeneic T cell populations per kg per dose per week to a human patient. In another specific implementation, administration includes administering approximately 2 x 10 6 One allogeneic T cell population per kg of T cells per week was administered to a human patient.

[0081] In some embodiments, the method of treating EBV-LPD described herein includes administering at least two doses of allogeneic T cell populations to a human patient. In specific embodiments, the method of treating EBV-LPD described herein includes administering 2, 3, 4, 5, or 6 doses of allogeneic T cell populations to a human patient.

[0082] In some embodiments, the method of treating EBV-LPD described herein includes a first cycle of administering one dose of allogeneic T-cell population weekly for three consecutive weeks, followed by an interval during which no dose of allogeneic T-cell population is administered, followed by a second cycle of administering one dose of allogeneic T-cell population weekly for three consecutive weeks. In some embodiments, the method of treating EBV-LPD described herein includes administering at least two cycles of administering one dose of allogeneic T-cell population weekly for three consecutive weeks, each cycle separated by an interval during which no dose of allogeneic T-cell population is administered. In specific embodiments, the method of treating EBV-LPD described herein includes administering 2, 3, 4, 5, or 6 cycles of administering one dose of allogeneic T-cell population weekly for three consecutive weeks, each cycle separated by an interval during which no dose of allogeneic T-cell population is administered. In one specific embodiment, the interval is approximately 3 weeks. Preferably, additional cycles are administered only if the preceding cycles have not shown toxicity (e.g., no grade 3-5 serious adverse events, classified according to NCI CTCAE 4.0).

[0083] In some embodiments, the first dosing regimen described herein is administered for a continuous first period, followed by a second period of the second and different dosing regimens described herein, wherein the first period is optionally separated by an interval (e.g., about 3 weeks). Preferably, the second dosing regimen is administered only if the first dosing regimen does not show toxicity (e.g., no grade 3-5 serious adverse events, according to NCITCAE 4.0 classification).

[0084] The term “approximately” should be interpreted as allowing for regular variation.

[0085] 1.5. Series of treatments using different T cell populations

[0086] In some embodiments, the method of treating EBV-LPD further includes administering a second allogeneic T-cell population comprising EBV-specific T cells to the human patient following administration of an allogeneic T-cell population; wherein the second allogeneic T-cell population is restricted by different HLA alleles shared with the cells of EBV-LPD. The second allogeneic T-cell population can be administered via any route and any dosage / administration regimen described in Section 4.4. In one specific embodiment, the method of treating EBV-LPD comprises administering one dose of the allogeneic T-cell population weekly for three consecutive weeks in a first cycle, followed by an interval during which no dose of the allogeneic T-cell population is administered, followed by a second cycle of one dose of the second allogeneic T-cell population weekly for three consecutive weeks. In yet another specific embodiment, the interval is approximately three weeks.

[0087] In some implementations, human patients do not respond, have an incomplete response, or a suboptimal response (i.e., human patients may still derive considerable benefit from continuous treatment, but the chance of achieving optimal long-term outcomes is reduced after administration of an allogeneic T-cell population and before administration of a second allogeneic T-cell population).

[0088] In a specific implementation scheme, two allogeneic EBV-specific T cell populations, each restricted by HLA alleles shared with EBV-LPD cells, are sequentially administered. In a specific implementation scheme, three allogeneic EBV-specific T cell populations, each restricted by HLA alleles shared with EBV-LPD cells, are sequentially administered. In a specific implementation scheme, four allogeneic EBV-specific T cell populations, each restricted by HLA alleles shared with EBV-LPD cells, are sequentially administered. In a specific implementation scheme, four or more allogeneic EBV-specific T cell populations, each restricted by HLA alleles shared with EBV-LPD cells, are sequentially administered.

[0089] 1.6. Patient

[0090] Human patients can be any patient who has EBV-LPD and has experienced unsuccessful combination chemotherapy (and in some embodiments, unsuccessful therapy with anti-CD20 monoclonal antibodies) and / or unsuccessful radiation therapy (and in some embodiments, unsuccessful therapy with anti-CD20 monoclonal antibodies).

[0091] LPD is a condition characterized by excessive proliferation of lymphocytes and can occur in immunocompromised patients. EBV-LPD that can be treated using the methods described herein includes, but is not limited to, B-cell hyperplasia, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), T-cell lymphoma, polymorphic or monomorphic EBV-LPD, EBV-positive Hodgkin lymphoma, Burkitt lymphoma, autoimmune lymphoproliferative syndromes, and mixed PTLD (post-transplant lymphoproliferative disorder). In one specific embodiment, EBV-LPD is EBV-positive lymphoma (e.g., EBV-positive B-cell lymphoma). In one specific embodiment, the EBV-LPD treated according to the methods described herein is present in the central nervous system of a human patient. In one specific embodiment, the EBV-LPD treated according to the methods described herein is present in the brain of a human patient.

[0092] In different implementations, the human patient was previously immunocompromised. In different implementations, the human patient was previously a transplant recipient. In some implementations, the human patient was previously a recipient of a solid organ transplant from a transplant donor. In some implementations, the human patient was previously a recipient of a multi-organ transplant (e.g., heart-lung transplant or kidney-pancreas transplant). Solid organ transplants may include, but are not limited to, kidney transplants, liver transplants, heart transplants, intestinal transplants, pancreas transplants, lung transplants, or combinations thereof. In one specific implementation, the solid organ transplant is a kidney transplant. In another specific implementation, the solid organ transplant is a liver transplant. In some implementations, the human patient was previously a recipient of a hematopoietic stem cell transplant (HSCT) from a transplant donor. HSCT can be a bone marrow transplant, peripheral blood stem cell transplant, or umbilical cord blood transplant. In a specific implementation, the allogeneic T cell population is derived from a donor other than the transplant donor. In other specific implementations, the allogeneic T cell population is derived from the transplant donor. In different implementations, the human patient was not previously a transplant recipient.

[0093] In the specific implementation plan, human patients are those infected with HIV.

[0094] In a specific implementation scheme, the human patient had previously received immunosuppressive therapy (e.g., after a solid organ transplant). In a particular aspect of this type of implementation scheme, the dose of immunosuppressive drug administered to the human patient was reduced, and the human patient experienced unsuccessful treatment for EBV-LPD with a reduced dose of immunosuppressive drug.

[0095] In a specific implementation plan, the human patient suffers from a primary immunodeficiency (e.g., a genetic condition that causes a primary immunodeficiency).

[0096] In other implementation schemes, the human patients are not immunocompromised.

[0097] 2. Example

[0098] This document provides certain embodiments illustrated by the following non-limiting examples, which demonstrate that therapies using allogeneic T cell populations comprising the EBV-specific T cells of the present invention are effective as low-toxicity second-line therapies in treating EBV-LPD that is resistant to combination chemotherapy or radiotherapy and also resistant to rituximab therapy.

[0099] 2.1. Example

[0100] Eleven recipients of solid organ transplants (SOTs) were referred to Memorial Sloan Kettering Cancer Center for the treatment of EBV-LPD, a form of lymphoma, following prior systemic chemotherapy. All had previously received rituximab and at least two prior combination chemotherapy regimens. Nine patients who received combination chemotherapy regimens and experienced incomplete response (3) or disease progression (6) were referred, along with two patients who relapsed after prior combination chemotherapy. Thus, all eleven patients had experienced unsuccessful combination chemotherapy for EBV-LPD.

[0101] If possible, evaluate lymphomas based on their source (SOT donor and host). If this is not possible, perform high-resolution HLA testing of SOT donor tissue against at least one allele to identify HLA-restricted strains in both the host and solid organ donor.

[0102] T cell lines were selected from an allogeneic T cell line library (each containing EBV-specific T cells) that shared at least 2 / 8 HLA alleles (A, B, C, and DR) with the patient and those restricted in EBV identification via alleles known to be expressed by lymphoma or by both host and solid organ donor tissues.

[0103] The patient received 2 x 10 6One T cell / kg / dose up to 3 weekly doses. If patients do not experience T-cell therapy-related toxicities (no grade 3-5 serious adverse events, graded according to NCI CTCAE 4.0) 5 weeks after starting therapy, they may receive additional cell cycles. Patients may receive subsequent cell cycles from different allogeneic T cell lines, preferably those restricted by different HLA alleles.

[0104] Some patients receive extra cycles of T cells, while others receive extra cycles of T cells from at least one different allogeneic T cell line restricted by different HLA alleles.

[0105] Of the 11 patients, 7 responded to the therapy. One responding patient received subsequent systemic chemotherapy for a relapse of low-grade disease in which she had not responded completely and had achieved partial remission after subsequent T-cell therapy. Two of the seven responding patients received subsequent rituximab therapy and EBV-specific T-cell therapy for retreatment. One patient with a complete response died from organ failure following prior cell therapy. Three patients with high disease burden who rapidly deteriorated at the start of therapy and progressed during the first cycle of T-cell therapy did not receive subsequent cycles of T-cell therapy.

[0106] In addition, a patient with CNS (central nervous system) involvement due to EBV-LPD who had an incomplete response to rituximab and radiation therapy alone was treated. This patient continued rituximab and began radiation therapy concurrently with his first cycle of EBV-specific T-cell therapy. EBV-LPD present in the brain (i.e., with brain involvement) is particularly difficult to treat with chemotherapy and radiation therapy because many chemotherapeutic agents cannot cross the blood-brain barrier, and radiation therapy often causes brain damage; however, the patient with brain involvement achieved partial remission. The first cycle of this T-cell therapy was graded NE (not evaluable), and he had a mild response at the end. After subsequent cycles, while receiving EBV-specific T-cell therapy as the sole therapy for EBV-LPD, he achieved substantial remission.

[0107] T-cell therapy has shown low toxicity.

[0108] Given that the allogeneic T cell population typically administered only lasts for a short period (generally shorter than that of hematopoietic stem cell transplant (HSCT) recipients) because the administered allogeneic T cells are rejected by the patient’s relatively intact immune system (as opposed to patients who are HSCT recipients), the fact that the method described in this article is effective in treating EBV-LPD in patients who were previously recipients of solid organ transplants is particularly unusual.

[0109] Table 1 below lists some of the treatment options the patient received.

[0110] Table 1. Treatment Plan

[0111]

[0112] 3. Merging by reference

[0113] This article cites various publications, the public content of which is incorporated into this article in its entirety through citation.

[0114] This disclosure relates to the following implementation plan.

[0115] 1. A method for treating a human patient with EBV-LPD (Ebola virus-associated lymphoproliferative disorder), the method comprising administering an allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the human patient has undergone unsuccessful combination chemotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by human leukocyte antigen (HLA) alleles common to cells with EBV-LPD.

[0116] 2. The method of implementation scheme 1, wherein the EBV-LPD is resistant to combination chemotherapy for EBV-LPD.

[0117] 3. The method of implementation scheme 1, wherein the human patient withdraws from combination chemotherapy due to poor tolerance of the combination chemotherapy.

[0118] 4. The method of any one of embodiments 1-3, wherein the combination chemotherapy includes a therapy with cyclophosphamide and prednisone.

[0119] 5. The method of implementation scheme 4, wherein the combination chemotherapy includes a low-dose cyclophosphamide and prednisone regimen.

[0120] 6. The method of any one of embodiments 1-3, wherein the combination chemotherapy comprises a therapy using cyclophosphamide and methylprednisolone.

[0121] 7. The method of implementation scheme 6, wherein the combination chemotherapy comprises a low-dose cyclophosphamide and methylprednisolone regimen.

[0122] 8. The method of any one of implementation schemes 1-3, wherein the human patient has undergone multiple different combination chemotherapy treatments for EBV-LPD without success.

[0123] 9. The method of implementation scheme 8, wherein the EBV-LPD is resistant to a variety of different combination chemotherapy therapies for treating EBV-LPD.

[0124] 10. The method of implementation scheme 8, wherein the human patient withdraws from multiple different combination chemotherapy therapies due to poor tolerance to multiple different combination chemotherapy therapies.

[0125] 11. The method of any one of embodiments 8-10, wherein at least one of the various combination chemotherapy therapies includes a therapy with cyclophosphamide and prednisone.

[0126] 12. The method of embodiment 11, wherein at least one of the various combination chemotherapy regimens comprises a low-dose cyclophosphamide and prednisone regimen.

[0127] 13. The method of any one of embodiments 8-10, wherein at least one of the various combination chemotherapy therapies includes a therapy with cyclophosphamide and methylprednisolone.

[0128] 14. The method of embodiment 13, wherein at least one of the various combination chemotherapy regimens comprises a low-dose cyclophosphamide and methylprednisolone regimen.

[0129] 15. The method of any one of embodiments 1-14, wherein the human patient has also failed to undergo radiotherapy for EBV-LPD.

[0130] 16. The method of embodiment 15, wherein the EBV-LPD is resistant to radiotherapy for EBV-LPD.

[0131] 17. The method of implementation scheme 15, wherein the human patient withdraws from radiotherapy due to poor tolerance to radiotherapy.

[0132] 18. A method for treating EBV-LPD in a human patient, the method comprising administering an allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the human patient has undergone unsuccessful radiotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by HLA alleles shared with cells of EBV-LPD.

[0133] 19. The method of embodiment 18, wherein the EBV-LPD is resistant to radiotherapy for EBV-LPD.

[0134] 20. The method of implementation scheme 18, wherein the human patient withdraws from radiotherapy due to poor tolerance to radiotherapy.

[0135] 21. The method of any one of embodiments 1-20, wherein EBV-LPD is also a B-cell lineage disease, and human patients have experienced unsuccessful treatment with anti-CD20 monoclonal antibodies for EBV-LPD.

[0136] 22. The method of embodiment 21, wherein the EBV-LPD is resistant to the therapy of treating EBV-LPD with an anti-CD20 monoclonal antibody.

[0137] 23. The method of implementation scheme 21, wherein the human patient withdraws from the therapy with the anti-CD20 monoclonal antibody due to poor tolerance to the therapy.

[0138] 24. The method of any one of embodiments 21-23, wherein the anti-CD20 monoclonal antibody is rituximab.

[0139] 25. The method of any one of embodiments 1-24, wherein the EBV-LPD is an EBV-positive lymphoma.

[0140] 26. The method of any one of embodiments 1-25, wherein the EBV-LPD is present in the central nervous system of a human patient.

[0141] 27. The method of implementation scheme 26, wherein the EBV-LPD is present in the brain of a human patient.

[0142] 28. The method of any one of embodiments 1-27, the method further comprising the step of determining at least one HLA allele of EBV-LPD cells by high-resolution typing prior to the administration step.

[0143] 29. The method of any one of embodiments 1-28, wherein the allogeneic T cell population and the EBV-LPD cells share at least 2 of the 8 HLA alleles.

[0144] 30. The method of embodiment 29, wherein the eight HLA alleles are two HLA alleles, two HLA-B alleles, two HLA-C alleles and two HLA-DR alleles.

[0145] 31. The method of any one of embodiments 1-30, wherein the EBV antigen recognized by the EBV-specific T cells is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, or LMP2.

[0146] 32. The method of any one of embodiments 1-31, wherein the human patient was a recipient of a solid organ transplant from a transplant donor.

[0147] 33. The method of implementation scheme 32, wherein the solid organ transplant is a kidney transplant, liver transplant, heart transplant, intestinal transplant, pancreas transplant, lung transplant, or a combination thereof.

[0148] 34. The method of implementation scheme 32, wherein the solid organ transplant is a kidney transplant.

[0149] 35. The method of implementation scheme 32, wherein the solid organ transplant is a liver transplant.

[0150] 36. The method of any one of embodiments 1-31, wherein the human patient was a recipient of a hematopoietic stem cell transplant from a transplant donor.

[0151] 37. The method of implementation scheme 36, wherein the hematopoietic stem cell transplantation is bone marrow transplantation, peripheral blood stem cell transplantation or umbilical cord blood transplantation.

[0152] 38. The method of any one of embodiments 32-37, wherein the allogeneic T cell population is derived from a donor other than the transplant donor.

[0153] 39. The method of any one of embodiments 1-38, the method further comprising the step of generating an allogeneic T cell population in vitro prior to the administration step.

[0154] 40. The method of embodiment 39, wherein the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells to one or more EBV antigens.

[0155] 41. The method of embodiment 40, wherein the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells using EBV-transformed B cells.

[0156] 42. The method of embodiment 40, wherein the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells with B cells transformed with EBV strain B95.8.

[0157] 43. The method of embodiment 40, wherein the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells using dendritic cells, cytokine-activated monocytes, or peripheral blood monocytes.

[0158] 44. The method of embodiment 43, wherein the step of sensitizing allogeneic T cells with dendritic cells, cytokine-activated monocytes or peripheral blood mononuclear cells comprises loading dendritic cells, cytokine-activated monocytes or peripheral blood mononuclear cells with at least one immunogenic peptide derived from one or more EBV antigens.

[0159] 45. The method of embodiment 43, wherein the step of sensitizing allogeneic T cells with dendritic cells, cytokine-activated monocytes or peripheral blood mononuclear cells comprises loading dendritic cells, cytokine-activated monocytes or peripheral blood mononuclear cells with an overlapping peptide library derived from one or more EBV antigens.

[0160] 46. ​​The method of embodiment 40, wherein the step of generating an allogeneic T cell population in vitro includes sensitizing the allogeneic T cells using artificial antigen-presenting cells (AAPC).

[0161] 47. The method of embodiment 46, wherein the step of sensitizing allogeneic T cells with AAPC includes loading AAPC with at least one immunogenic peptide derived from one or more EBV antigens.

[0162] 48. The method of embodiment 46, wherein the step of sensitizing allogeneic T cells with AAPC includes loading AAPC with an overlapping peptide library derived from one or more EBV antigens.

[0163] 49. The method of embodiment 46, wherein the step of sensitizing allogeneic T cells with AAPC includes engineering AAPC to express at least one immunogenic EBV peptide or protein in AAPC.

[0164] 50. The method of any one of embodiments 40-49, wherein the method further comprises cryopreserving allogeneic T cells after sensitization.

[0165] 51. The method of any one of embodiments 1-50, the method further comprising, prior to the administration step, thawing cryopreserved EBV-antigen-sensitized allogeneic T cells and expanding the allogeneic T cells in vitro to generate an allogeneic T cell population.

[0166] 52. The method of any one of embodiments 1-51, the method further comprising, prior to the administration step, thawing the cryopreserved allogeneic T cell population.

[0167] 53. The method of any one of embodiments 1-49, wherein the allogeneic T cell population is derived from a T cell line.

[0168] 54. The method of embodiment 53, the method further comprising the step of selecting a T cell line from a variety of cryopreserved T cell line libraries prior to the administration step.

[0169] 55. The method of embodiment 53 or 54, the method further comprising, prior to the administration step, thawing the cryopreserved T cell line.

[0170] 56. The method of any one of embodiments 53-55, wherein the method further comprises, prior to the administration step, an in vitro expansion of the T cell line.

[0171] 57. The method of any one of embodiments 1-56, wherein the administration is performed by infusion of an allogeneic T cell population.

[0172] 58. The method of implementation scheme 57, wherein the infusion is an intravenous bolus injection.

[0173] 59. The method of any one of embodiments 1-58, wherein the giving comprises giving at least about 1 x 10 5 One allogeneic T cell population per kg of T cells per week was administered to a human patient.

[0174] 60. The method of any one of embodiments 1-58, wherein the giving comprises giving approximately 1 x 10 6 - Approximately 2 x 10 6 One allogeneic T cell population per kg of T cells per week was administered to a human patient.

[0175] 61. The method of any one of embodiments 1-58, wherein the giving comprises giving approximately 1 x 10 6 One allogeneic T cell population per kg of T cells per week was administered to a human patient.

[0176] 62. The method of any one of embodiments 1-58, wherein the giving comprises placing approximately 2 x 10 6 One allogeneic T cell population per kg of T cells per week was administered to a human patient.

[0177] 63. The method of any one of embodiments 1-62, wherein the administration comprises administering at least two doses of allogeneic T cell populations to a human patient.

[0178] 64. The method of implementation scheme 63, wherein the administration comprises administering 2, 3, 4, 5 or 6 doses of allogeneic T cell populations to a human patient.

[0179] 65. The method of any one of embodiments 1-62, wherein the administration comprises a first cycle of administering one dose of allogeneic T cell population once a week for three consecutive weeks, followed by an interval during which no dose of allogeneic T cell population is administered, followed by a second cycle of administering one dose of allogeneic T cell population once a week for three consecutive weeks.

[0180] 66. The method of any one of embodiments 1-62, wherein the administration comprises administering an allogeneic T cell population once a week for three consecutive weeks, for 2, 3, 4, 5 or 6 cycles, each cycle being separated by an interval during which no dose of the allogeneic T cell population is administered.

[0181] 67. The method of implementation scheme 65 or 66, wherein the interval period is approximately 3 weeks.

[0182] 68. The method of any one of embodiments 1-67, the method further comprising, after administering an allogeneic T cell population to a human patient, administering a second allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the second allogeneic T cell population is restricted by different HLA alleles shared with the cells of EBV-LPD.

[0183] 69. The method of embodiment 68, wherein the administration comprises administering one dose of allogeneic T cell population once a week for three consecutive weeks in a first cycle, followed by an interval during which no dose of allogeneic T cell population is administered, followed by one dose of a second allogeneic T cell population once a week for three consecutive weeks in a second cycle.

[0184] 70. The method of implementation scheme 69, wherein the interval period is approximately 3 weeks.

[0185] 71. The method of any one of embodiments 68-70, wherein the human patient has no response, incomplete response, or suboptimal response after administration of an allogeneic T cell population and before administration of a second allogeneic T cell population.

Claims

1. A method for treating a human patient with EBV-LPD (Ebola virus-associated lymphoproliferative disorder), the method comprising administering an allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the human patient has undergone unsuccessful combination chemotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by human leukocyte antigen (HLA) alleles common to cells with EBV-LPD.

2. The method of claim 1, wherein the EBV-LPD is resistant to combination chemotherapy for EBV-LPD.

3. A method for treating EBV-LPD in a human patient, the method comprising administering an allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the human patient has undergone unsuccessful radiotherapy for EBV-LPD, and wherein the allogeneic T cell population is restricted by HLA alleles shared with cells of EBV-LPD.

4. The method of any one of claims 1-3, wherein the administration comprises a first cycle of administering one dose of allogeneic T cell population per week for three consecutive weeks, followed by an interval during which no dose of allogeneic T cell population is administered, followed by a second cycle of administering one dose of allogeneic T cell population per week for three consecutive weeks.

5. The method of any one of claims 1-4, wherein the administration comprises administering an allogeneic T cell population once a week for three consecutive weeks, for 2, 3, 4, 5, or 6 cycles, each cycle being separated by an interval during which no dose of the allogeneic T cell population is administered.

6. The method of claim 4 or 5, wherein the interval period is approximately 3 weeks.

7. The method of any one of claims 1-6, the method further comprising, after administering an allogeneic T cell population to a human patient, administering a second allogeneic T cell population comprising EBV-specific T cells to the human patient; wherein the second allogeneic T cell population is restricted by different HLA alleles shared with the cells of EBV-LPD.

8. The method of claim 7, wherein the administration comprises a first cycle of administering one dose of allogeneic T cell population per week for three consecutive weeks, followed by an interval during which no dose of allogeneic T cell population is administered, followed by a second cycle of administering one dose of second allogeneic T cell population per week for three consecutive weeks.

9. The method of claim 8, wherein the interval is approximately 3 weeks.

10. The method of any one of claims 7-9, wherein the human patient has no response, incomplete response, or suboptimal response after administration of the allogeneic T cell population and before administration of the second allogeneic T cell population.

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