T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first genemodified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/ LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

La thérapie par cellules T à récepteur d'antigène chimérique (CAR) a produit d'incroyables réponses cliniques contre plusieurs tumeurs malignes, mais elle laisse une marge de manœuvre pour l'échappement de l'antigène. Une nouvelle approche pour surmonter ce problème consisterait à combiner la capacité des CAR à cibler les tumeurs avec la capacité des cellules dendritiques (CD) à amorcer les cellules T afin de générer une thérapie cellulaire qui favorise la propagation de l'épitope plutôt que la destruction directe de la cible. J'ai donc émis l'hypothèse que les cellules dendritiques exprimant les CAR (CAR-CD) peuvent renforcer l'amorçage des cellules T contre les cibles tumorales afin de produire un contrôle adaptatif des tumeurs médié par les cellules T. En utilisant des CD dérivées de la moelle osseuse murine, j'ai d'abord développé un nouveau protocole pour générer des CD. En ajoutant de l'IFNα aux cultures de DC GM-CSF, j'ai généré des CD qui expriment des niveaux plus élevés de molécules stimulant les cellules T et qui induisent une plus forte prolifération des cellules T CD8+ in-vitro par rapport aux CD générées avec le GM-CSF seul. In vivo, ces CD favorisent des réponses effectrices plus fortes, les cellules T CD8+ résultantes exprimant des niveaux plus élevés de marqueurs effectrices, notamment KLRG1 et TIM3, mais des niveaux plus faibles de molécules inhibitrices, notamment PD-1 et CD73. L'expression d'un CAR dans ces DC leur permet de tuer directement des cibles tumorales et d'acquérir des antigènes tumoraux. Plus important encore, ces CAR-CD sont en synergie avec les cellules T CD8+ pour contrôler les cellules tumorales in-vitro, en améliorant la prolifération et la capacité de destruction des cellules T. Chez la souris, les CAR-CD agissent comme un vaccin in vivo en favorisant la génération de réponses de cellules T spécifiques de la tumeur lorsqu'elles sont injectées par voie intratumorale, ce qui permet d'améliorer le contrôle de la tumeur. / Chimeric Antigen Receptor (CAR) T cell therapy has produced unbelievable clinical responses against several malignancies however, this therapy leaves room for antigen escape. One novel approach to overcome this would be to combine the tumor targeting ability of CARs with the T cell priming capacity of dendritic cells (DCs) to generate a cell therapy that provokes endogenous adaptive immunity through epitope spreading rather than just direct target killing. Therefore, I hypothesized that CAR expressing DCs (CAR-DCs) can enhance T cell priming against tumor targets to produce adaptive T cell mediated tumor control. Using murine bone marrow derived DCs, I first developed a new protocol for generating DCs using IFNα. The addition of IFNα to GM-CSF DC cultures generated DCs that express higher levels of T cell stimulatory molecules and induce stronger CD8+ T cell proliferation in-vitro compared to DCs generated with GM-CSF alone. In-vivo, these DCs promote stronger effector responses with the resulting CD8+ T cells expressing higher levels of effector markers including KLRG1 and TIM3 but lower levels of inhibitory molecules including PD-1 and CD73. The expression of a CAR in these DCs allowed them to directly kill tumor targets and acquire tumor antigens. More importantly, these CAR-DCs synergized with CD8+ T cells to control tumor cells in-vitro, enhancing the proliferation and killing capacity of the T cells. In mice, CAR-DCs act as an in-vivo vaccine promoting the generation of tumor specific T cell responses when injected intratumorally, producing enhanced tumor control.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CART cell manufacturing process.

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In the last decade, six CAR T cell therapies against hematological malignancies have been approved for commercial manufacturing and several clinical trials are underway. This has led to extensive preclinical research focused on optimizing individual manufacturing steps of adoptive T cell therapeutics. Ex vivo expansion of T cells is one of the crucial manufacturing steps, as is necessary to obtain clinically required cell numbers for infusion. However, ex vivo expansion is also a complex step as it involves multiple different variables including culture medium, serum and cytokine supplementation, activation reagent and mode of genetic modification. Consequently, our understanding of changes in T cells during ex vivo expansion and the impact of expansion conditions on the final product; and thus the outcome of the therapy remain mostly elusive. Therefore, this project was designed to understand the changes in T cells at different stages of ex vivo expansion compared to freshly isolated T cells with a focus on understanding the ongoing transcriptional changes. The T cells were isolated from healthy blood donor buffy coats using FABian®-Cell Isolation System based on Fab-TACS technology. T cells were cultured for 7 days in X-VIVO 15 media (supplemented with 5% human serum and 50 IU/ml IL-2) with activation for initial 3 days using anti-CD3/CD28 TranAct. The T cell kinetics during ex vivo expansion was characterized based on cell activation, differentiation and proliferation. For whole transcriptome sequencing, Total RNA was harvested from 6 different time points, freshly isolated cells (0 hr), and cells cultured for 4, 12, 24, 72 and 168 hr (7 days). The RNA sequencing libraries were prepared using “Illumina TruSeq Stranded Total RNA library prep' workflow and whole transcriptome sequencing was performed on Illumina Novaseq 6000. Further, changes in T cell trafficking capabilities, cell size and cell cycle progression were studied in freshly isolated T cells and cultured cells. The changes in T cell trafficking was studied by analyzing the changes in VLA4 mediated T cell adhesion to VCAM1 coated surface under increasing shear stress. The cell size and volume of freshly isolated T cells and cultured cells were analyzed using multisizer instrument. Additionally, an in vitro model was developed to simulate the behavior of cultured T cells upon re-infusion into the blood and changes in cell cycle was analyzed. The components of in vitro reconstituted blood model were pooled human AB serum, erythrocyte concentrates and cultured T cells. The absolute lymphocyte count in buffy coats and total number of T cells isolated per buffy coat were in range compared to cell isolation and enrichment through standard leukapheresis. Thus suggesting that healthy donor-derived buffy coats and enrichment of T cells using Fab-TACS technology can be a suitable starting material and cell enrichment device respectively. The T cell growth kinetics was analyzed based on surface expression of specific markers, which also closely resembled their gene expression. The T cell kinetics observed during ex vivo expansion was similar to T cell kinetics observed in several preclinical CAR T cell expansion studies. The T cell proliferation in terms of increase in cell numbers and gene ontology (GO) terms related to DNA replication and cell division were significantly enriched only after 3 days of ex vivo expansion. The final cell numbers after 7 days of ex vivo expansion were approx. 1.0E+9 T cells, which was well above the clinically required infusion dosage of currently approved CAR T cell therapies. Taken together, the ex vivo expansion protocol followed in this study generates T cells in range required for clinical infusion dose and the growth kinetics of T cells observed were in line with the commercial expansion protocol. Hierarchical clustering of genes based on their expression over time identified 29 different gene-clusters which followed the pattern of mono-, bi- and triphasic modulation. The gene-clusters 11 and 18 were significantly enriched with T cell immune function related GO terms. The GO analysis of differentially expressed genes identified enrichment several bioprocesses, signaling pathways and T cell immune functions including commonly known activation, differentiation and proliferation. The ex vivo expansion of T cells was associated with early (i.e. upto 24 hr time point) enrichment of several GO terms associated with cytokine production such as IL-1, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17, TNF and IFN-γ. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Mitogen-activated protein kinase (MAPK) signaling cascades related GO terms were enriched as a result of autocrine signaling mediated by cytokines released during ex vivo expansion. These data demonstrate that cytokine release in T cells is activated during ex vivo manufacturing, and should be considered during future optimization. The gene expression analysis of commonly known exhaustion markers revealed two different patterns of expression. The CTLA4, TIGIT, TBX21 and BATF was upregulated at early time points. Whereas, the expression of TIM3, LAG3 and CX3CR1 was upregulated at later time points. The early expression of exhaustion markers can be attributed to immune check point function to prevent over-activation, and later expression of exhaustion markers may contribute to inhibitory function. In vitro investigation of ex vivo expanded T cells exhibited stronger VLA4 mediated adhesion to VCAM1 coated surface compared to freshly isolated cells under increasing shear stress. This was in contrast to the downregulation of alpha 4/beta 1 integrin gene expression during ex vivo expansion. The cell size analysis revealed cultured T cells were larger in terms of both size and volume at the end of 7-day culture period with doubled cell volume compared to freshly isolated T cells. These results taken together, suggest that increased adhesion capacity and increased cell size, after T cell expansion may be associated with accumulation of T cells in lungs upon infusion. The freshly isolated T cells that closely represent the T cells circulating in peripheral blood were arrested in G0/G1 phase. However, during ex vivo expansion T cells entered cell cycle, and T cells were found to be predominantly in S+G2/M phase on day 3, 5 and 7. Surprisingly, the cultured T cells were still in cell cycle even after 48 h of incubation in reconstituted blood in vitro. This suggests that a prolonged resting phase of ex vivo expanded T cells for more 48 hr before infusion into the patients can be advantageous in minimizing the risks associated with T cell therapy. In conclusion, this study has revealed a number of novel insights into transcriptional regulation and signaling processes occurring during culture expansion. In the study, the different patterns of transcriptional regulation and enrichment of various associated bio-processes and signaling pathways during ex vivo expansion were explored. In addition, an in-depth analysis of genes related to T cell activation and differentiation, adhesion and migration, and exhaustion markers was performed. The protein-protein interaction analysis and transcriptional factor enrichment analysis provide valuable data for further in silico investigations of transcriptional changes in T cells during ex vivo expansion. Additionally, this study provides a comprehensive overview of long non-coding RNAs at different stages of ex vivo expansion of T cells, thus providing a resource for novel understanding of impact of lncRNAs on T cells during ex vivo expansion for adoptive T cell therapies. The complete data of 48 transcriptomes derived from 8 donors over 6 time points is reposited (GEO: GSE250311) to a publicly available database and will allow exploration for future studies which aim at the characterization of alterations in expanded T cells for therapy, and optimization of conditions for their future use in patients.

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

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Ex vivo expansion of T lymphocytes is a central process in the generation of cellular therapies targeted at tumors and other disease-relevant structures,which currently cannot be reached by established pharmaceuticals. The influence of culture conditions on T cell functions is, however, incompletely understood. In clinical applications of ex vivo expanded T cells, so far, a relatively classical standard cell culture methodology has been established. The expanded cells have been characterized in both preclinical models and clinical studies mainly using a therapeutic endpoint, for example antitumor response and cytotoxic function against cellular targets, whereas the influence of manipulations of T cells ex vivo including transduction and culture expansion has been studied to a much lesser detail, or in many contexts remains unknown. This includes the circulation behavior of expanded T cells after intravenous application, their intracellular metabolism and signal transduction, and their cytoskeletal (re)organization or their adhesion, migration, and subsequent intra-tissue differentiation. This review aims to provide an overview of established T cell expansion methodologies and address unanswered questions relating in vivo interaction of ex vivo expanded T cells for cellular therapy.