Towards GMP production of γðTCR engineered T cells

Dissertação de Mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A leucemia mieloide aguda (AML) é uma das mais frequentes doenças malignas hematológicas descritas, e é caracterizada como uma anomalia heterogénea clonal de células progenitoras do sistema hemapoiético. Assim, células progenitoras hematopoiéticas (também designadas por “blasts”) perdem descontroladamente a habilidade de se diferenciarem normalmente, incapacitando-as de responder a reguladores de proliferação. Consequentemente, depois do diagnóstico inicial, hemorragias, infecções, ou infiltrações em órgãos podem ser fatais na ausência de tratamento. Ao longo dos anos, enquanto o uso de quimioterapia falhava em demonstrar eficácia em doentes que sofrem de AML, a imunoterapia surgiu como uma das estratégias mais promissoras para tratamento destes pacientes. Usando diversas estratégias, o transplante alogénico de células estaminais surgiu como tratamento de primeira linha. No entanto, a rejeição do transplante pelo recipiente (GVHD) ou a recaída de AML acarretam riscos fatais em pacientes que foram submetidos a este transplante. Com o intuito de fornecer eficácia anti tumoral e uma proteção duradoura, várias abordagens foram empregues, como a vacinação de células dendríticas ou infusão linfocítica (DLI). Porém, esta última não permite um controlo tumoral, impulsionando esforços na procura de soluções mais eficientes. Atualmente, avanços na engenharia genética estabeleceram uma plataforma que permite a transferência genética para células T de recetores de células T (TCRs) com alta reatividade tumoral. O uso de receptores de célulasT (TCRse recetores de antigénios quiméricos (CARS) demonstram limitações devido a restrição HLA, toxicidade “off-target” e imunogenicidade. Consequentemente, uma maior atenção tem-se dado a certos tipos de células T e aos seus recetores(TCRs), devido ao reconhecimento de células em stress (células tumorais) não restrito pelo sistema antígeno leucocitário humano (HLA). Num contexto de terapia celular adotiva (ACT), este recetor de imunidade inata/adaptiva pode ser usado em células T para matar células tumorais sem as limitações dos TCRs e dos CARs, algo demonstrado pelo nosso grupo. Nesta tese, centrámos os nossos objetivos no sentido de desenvolver um protocolo de boas práticas de fabricação (GMP) para a produção de células T geneticamente modificadas com o um recetor de células T altamente reativas a células de leucemia mieloide aguda. Para este fim, iremos otimizar diversos passos de um protocolo de investigação de transdução retroviral. Neste projeto, inserimos um V9V2 TCR clone 5 em células T estimuladas com anti-CD3/CD28 beads. Depois da expansão com IL- 7 e IL-15, as células T geneticamente modificadas foram separadas de células T pouco ou não modificadas, usando um novo passo de depleção de células que expressam TCRs. Como resultado fomos capazes de gerar uma grande população de células T geneticamente modificadas com TCR, memoria efetoras e efetoras como estado de diferenciação fenotípica. Sobretudo, quando este produto celular foi testado contra amostras tumorais primarias de AML, estas células T geneticamente modificadas com TCR libertaram INF-. O sucesso no escalonamento da produção destas células e o novo passo de purificação iii destas, garantem uma fácil transição para a clinica, permitindo o seu uso em ensaios clinicos de fase I em pacientes de AML de baixo risco que sofreram um transplante alogénico de células estaminais (allo-SCT).Acute Myeloid Leukemia (AML) is one of the most frequent hematological malignancy within those described to date. It is characterized as a heterogeneous clonal aberrance of progenitor cells of the human hematopoietic system. Hereby, these hematopoietic progenitor cells (“blasts”) disorderly lose the ability to undergo a normal differentiation process consequently exerting a poor response to regular proliferative regulators. Therefore, after an initial diagnosis, fatal infections, organ infiltration and bleeding are consequences of this loss in the absence of treatment. Throughout the years, whereas chemotherapy regimens fail to prove efficacy to accelerate and lengthen the remission state in patients suffering from AML, immunotherapy surfaced as one of the most promising AML therapeutic approaches. As several allogeneic-stem cell transplantation (allo-SCT) strategies remain first line treatments available for AML patients, graft-versus-host-disease (GVHD) or the relapse of the AML patient become major life-threatening complications post-allo-SCT. To deliver antitumor efficacy and long-term protection multiple approaches have been employed, such as dendritic cell (DC) vaccination or donor lymphocyte infusions (DLI). Nonetheless, this latter does not provide proper tumor control, driving efforts to seek more efficient solutions. Currently, genetic engineering has evolved to provide a platform to transfer highly tumor reactive T cell receptors (TCRs) into T cells. The use of tumor specific T cell receptors (Acute Myeloid Leukemia (AML) is one of the most frequent hematological malignancy within those described to date. It is characterized as a heterogeneous clonal aberrance of progenitor cells of the human hematopoietic system. Hereby, these hematopoietic progenitor cells (“blasts”) disorderly lose the ability to undergo a normal differentiation process consequently exerting a poor response to regular proliferative regulators. Therefore, after an initial diagnosis, fatal infections, organ infiltration and bleeding are consequences of this loss in the absence of treatment. Throughout the years, whereas chemotherapy regimens fail to prove efficacy to accelerate and lengthen the remission state in patients suffering from AML, immunotherapy surfaced as one of the most promising AML therapeutic approaches. As several allogeneic-stem cell transplantation (allo-SCT) strategies remain first line treatments available for AML patients, graft-versus-host-disease (GVHD) or the relapse of the AML patient become major life-threatening complications post-allo-SCT. To deliver antitumor efficacy and long-term protection multiple approaches have been employed, such as dendritic cell (DC) vaccination or donor lymphocyte infusions (DLI). Nonetheless, this latter does not provide proper tumor control, driving efforts to seek more efficient solutions. Currently, genetic engineering has evolved to provide a platform to transfer highly tumor reactive T cell receptors (TCRs) into T cells. The use of tumor specific T cell receptors (TCRs) and chimeric antigen receptors (CARs) show their limitations due to HLA-restriction anti-tumor activity, off-target reactivity and immunogenicity. As a consequence, certain subsets of T cells and their receptors (TCRs), have arisen attention due to their non-HLA restricted recognition of stressed cells (tumor cells). In the context of adoptive T cell therapy (ACT), this innate-like receptor can be used in T cells to deliver anti-tumor activity in tumor cells without the limitations of TCRs or CARs, as demonstrated by our group. In this thesis, we set our aims towards developing a good manufacturing production (GMP) protocol for the production of AML-reactive TCR engineered T cells by optimizing several steps of a research-grade retroviral transduction protocol. We inserted a highly tumor reactive V9V2 TCR derived from clone 5 in anti-CD3/CD28 bead stimulated T cells. Following expansion with IL-7 and IL-15, engineered T cells were purified from poorly/non-engineered T cells using a novel TCR depletion step. As a result, we were able to generate a major population of highly purified TCR engineered T cells in an effector memory and effector differentiation phenotype. More importantly, when challenged against AML primary tumor samples, this engineered T cell product is able release INF-. The successful upscaling of the manufacturing process of this genetically modified T cell product using a novel purifying method, grants an easy translation for a clinical-grade setting enabling its use in a phase I clinical trial on poor/low risk AML patients that have undergone allogeneic – stem cell transplantation (allo-SCT).TCRs) and chimeric antigen receptors (CARs) show their limitations due to HLA-restriction anti-tumor activity, off-target reactivity and immunogenicity. As a consequence, certain subsets of Acute Myeloid Leukemia (AML) is one of the most frequent hematological malignancy within those described to date. It is characterized as a heterogeneous clonal aberrance of progenitor cells of the human hematopoietic system. Hereby, these hematopoietic progenitor cells (“blasts”) disorderly lose the ability to undergo a normal differentiation process consequently exerting a poor response to regular proliferative regulators. Therefore, after an initial diagnosis, fatal infections, organ infiltration and bleeding are consequences of this loss in the absence of treatment. Throughout the years, whereas chemotherapy regimens fail to prove efficacy to accelerate and lengthen the remission state in patients suffering from AML, immunotherapy surfaced as one of the most promising AML therapeutic approaches. As several allogeneic-stem cell transplantation (allo-SCT) strategies remain first line treatments available for AML patients, graft-versus-host-disease (GVHD) or the relapse of the AML patient become major life-threatening complications post-allo-SCT. To deliver antitumor efficacy and long-term protection multiple approaches have been employed, such as dendritic cell (DC) vaccination or donor lymphocyte infusions (DLI). Nonetheless, this latter does not provide proper tumor control, driving efforts to seek more efficient solutions. Currently, genetic engineering has evolved to provide a platform to transfer highly tumor reactive T cell receptors (TCRs) into T cells. The use of tumor specific T cell receptors (TCRs) and chimeric antigen receptors (CARs) show their limitations due to HLA-restriction anti-tumor activity, off-target reactivity and immunogenicity. As a consequence, certain subsets of T cells and their receptors (TCRs), have arisen attention due to their non-HLA restricted recognition of stressed cells (tumor cells). In the context of adoptive T cell therapy (ACT), this innate-like receptor can be used in T cells to deliver anti-tumor activity in tumor cells without the limitations of TCRs or CARs, as demonstrated by our group. In this thesis, we set our aims towards developing a good manufacturing production (GMP) protocol for the production of AML-reactive TCR engineered T cells by optimizing several steps of a research-grade retroviral transduction protocol. We inserted a highly tumor reactive V9V2 TCR derived from clone 5 in anti-CD3/CD28 bead stimulated T cells. Following expansion with IL-7 and IL-15, engineered T cells were purified from poorly/non-engineered T cells using a novel TCR depletion step. As a result, we were able to generate a major population of highly purified TCR engineered T cells in an effector memory and effector differentiation phenotype. More importantly, when challenged against AML primary tumor samples, this engineered T cell product is able release INF-. The successful upscaling of the manufacturing process of this genetically modified T cell product using a novel purifying method, grants an easy translation for a clinical-grade setting enabling its use in a phase I clinical trial on poor/low risk AML patients that have undergone allogeneic – stem cell transplantation (allo-SCT). T cells and their receptors (TCRs), have arisen attention due to their non-HLA restricted recognition of stressed cells (tumor cells). In the context of adoptive T cell therapy (ACT), this innate-like receptor can be used in Acute Myeloid Leukemia (AML) is one of the most frequent hematological malignancy within those described to date. It is characterized as a heterogeneous clonal aberrance of progenitor cells of the human hematopoietic system. Hereby, these hematopoietic progenitor cells (“blasts”) disorderly lose the ability to undergo a normal differentiation process consequently exerting a poor response to regular proliferative regulators. Therefore, after an initial diagnosis, fatal infections, organ infiltration and bleeding are consequences of this loss in the absence of treatment. Throughout the years, whereas chemotherapy regimens fail to prove efficacy to accelerate and lengthen the remission state in patients suffering from AML, immunotherapy surfaced as one of the most promising AML therapeutic approaches. As several allogeneic-stem cell transplantation (allo-SCT) strategies remain first line treatments available for AML patients, graft-versus-host-disease (GVHD) or the relapse of the AML patient become major life-threatening complications post-allo-SCT. To deliver antitumor efficacy and long-term protection multiple approaches have been employed, such as dendritic cell (DC) vaccination or donor lymphocyte infusions (DLI). Nonetheless, this latter does not provide proper tumor control, driving efforts to seek more efficient solutions. Currently, genetic engineering has evolved to provide a platform to transfer highly tumor reactive T cell receptors (TCRs) into T cells. The use of tumor specific T cell receptors (TCRs) and chimeric antigen receptors (CARs) show their limitations due to HLA-restriction anti-tumor activity, off-target reactivity and immunogenicity. As a consequence, certain subsets of T cells and their receptors (TCRs), have arisen attention due to their non-HLA restricted recognition of stressed cells (tumor cells). In the context of adoptive T cell therapy (ACT), this innate-like receptor can be used in T cells to deliver anti-tumor activity in tumor cells without the limitations of TCRs or CARs, as demonstrated by our group. In this thesis, we set our aims towards developing a good manufacturing production (GMP) protocol for the production of AML-reactive TCR engineered T cells by optimizing several steps of a research-grade retroviral transduction protocol. We inserted a highly tumor reactive V9V2 TCR derived from clone 5 in anti-CD3/CD28 bead stimulated T cells. Following expansion with IL-7 and IL-15, engineered T cells were purified from poorly/non-engineered T cells using a novel TCR depletion step. As a result, we were able to generate a major population of highly purified TCR engineered T cells in an effector memory and effector differentiation phenotype. More importantly, when challenged against AML primary tumor samples, this engineered T cell product is able release INF-. The successful upscaling of the manufacturing process of this genetically modified T cell product using a novel purifying method, grants an easy translation for a clinical-grade setting enabling its use in a phase I clinical trial on poor/low risk AML patients that have undergone allogeneic – stem cell transplantation (allo-SCT). T cells to deliver anti-tumor activity in tumor cells without the limitations of Acute Myeloid Leukemia (AML) is one of the most frequent hematological malignancy within those described to date. It is characterized as a heterogeneous clonal aberrance of progenitor cells of the human hematopoietic system. Hereby, these hematopoietic progenitor cells (“blasts”) disorderly lose the ability to undergo a normal differentiation process consequently exerting a poor response to regular proliferative regulators. Therefore, after an initial diagnosis, fatal infections, organ infiltration and bleeding are consequences of this loss in the absence of treatment. Throughout the years, whereas chemotherapy regimens fail to prove efficacy to accelerate and lengthen the remission state in patients suffering from AML, immunotherapy surfaced as one of the most promising AML therapeutic approaches. As several allogeneic-stem cell transplantation (allo-SCT) strategies remain first line treatments available for AML patients, graft-versus-host-disease (GVHD) or the relapse of the AML patient become major life-threatening complications post-allo-SCT. To deliver antitumor efficacy and long-term protection multiple approaches have been employed, such as dendritic cell (DC) vaccination or donor lymphocyte infusions (DLI). Nonetheless, this latter does not provide proper tumor control, driving efforts to seek more efficient solutions. Currently, genetic engineering has evolved to provide a platform to transfer highly tumor reactive T cell receptors (TCRs) into T cells. The use of tumor specific T cell receptors (TCRs) and chimeric antigen receptors (CARs) show their limitations due to HLA-restriction anti-tumor activity, off-target reactivity and immunogenicity. As a consequence, certain subsets of T cells and their receptors (TCRs), have arisen attention due to their non-HLA restricted recognition of stressed cells (tumor cells). In the context of adoptive T cell therapy (ACT), this innate-like receptor can be used in T cells to deliver anti-tumor activity in tumor cells without the limitations of TCRs or CARs, as demonstrated by our group. In this thesis, we set our aims towards developing a good manufacturing production (GMP) protocol for the production of AML-reactive TCR engineered T cells by optimizing several steps of a research-grade retroviral transduction protocol. We inserted a highly tumor reactive V9V2 TCR derived from clone 5 in anti-CD3/CD28 bead stimulated T cells. Following expansion with IL-7 and IL-15, engineered T cells were purified from poorly/non-engineered T cells using a novel TCR depletion step. As a result, we were able to generate a major population of highly purified TCR engineered T cells in an effector memory and effector differentiation phenotype. More importantly, when challenged against AML primary tumor samples, this engineered T cell product is able release INF-. The successful upscaling of the manufacturing process of this genetically modified T cell product using a novel purifying method, grants an easy translation for a clinical-grade setting enabling its use in a phase I clinical trial on poor/low risk AML patients that have undergone allogeneic – stem cell transplantation (allo-SCT).TCRs or CARs, as demonstrated by our group. In this thesis, we set our aims towards developing a good manufacturing production (GMP) protocol for the production of AML-reactive Acute Myeloid Leukemia (AML) is one of the most frequent hematological malignancy within those described to date. It is characterized as a heterogeneous clonal aberrance of progenitor cells of the human hematopoietic system. Hereby, these hematopoietic progenitor cells (“blasts”) disorderly lose the ability to undergo a normal differentiation process consequently exerting a poor response to regular proliferative regulators. Therefore, after an initial diagnosis, fatal infections, organ infiltration and bleeding are consequences of this loss in the absence of treatment. Throughout the years, whereas chemotherapy regimens fail to prove efficacy to accelerate and lengthen the remission state in patients suffering from AML, immunotherapy surfaced as one of the most promising AML therapeutic approaches. As several allogeneic-stem cell transplantation (allo-SCT) strategies remain first line treatments available for AML patients, graft-versus-host-disease (GVHD) or the relapse of the AML patient become major life-threatening complications post-allo-SCT. To deliver antitumor efficacy and long-term protection multiple approaches have been employed, such as dendritic cell (DC) vaccination or donor lymphocyte infusions (DLI). Nonetheless, this latter does not provide proper tumor control, driving efforts to seek more efficient solutions. Currently, genetic engineering has evolved to provide a platform to transfer highly tumor reactive T cell receptors (TCRs) into T cells. The use of tumor specific T cell receptors (TCRs) and chimeric antigen receptors (CARs) show their limitations due to HLA-restriction anti-tumor activity, off-target reactivity and immunogenicity. As a consequence, certain subsets of T cells and their receptors (TCRs), have arisen attention due to their non-HLA restricted recognition of stressed cells (tumor cells). In the context of adoptive T cell therapy (ACT), this innate-like receptor can be used in T cells to deliver anti-tumor activity in tumor cells without the limitations of TCRs or CARs, as demonstrated by our group. In this thesis, we set our aims towards developing a good manufacturing production (GMP) protocol for the production of AML-reactive TCR engineered T cells by optimizing several steps of a research-grade retroviral transduction protocol. We inserted a highly tumor reactive V9V2 TCR derived from clone 5 in anti-CD3/CD28 bead stimulated T cells. Following expansion with IL-7 and IL-15, engineered T cells were purified from poorly/non-engineered T cells using a novel TCR depletion step. As a result, we were able to generate a major population of highly purified TCR engineered T cells in an effector memory and effector differentiation phenotype. More importantly, when challenged against AML primary tumor samples, this engineered T cell product is able release INF-. The successful upscaling of the manufacturing process of this genetically modified T cell product using a novel purifying method, grants an easy translation for a clinical-grade setting enabling its use in a phase I clinical trial on poor/low risk AML patients t