Externally-Controlled Systems for Immunotherapy: From Bench to Bedside

We thank GENYO Institute and LentiStem Biotech for the support to compile of the necessary information to write this review. We also thank Fundación Poco Frecuente (FPF) and Asociación Española de Enfermos con Glucogenosis (AEEG) for their kindly support.Immunotherapy is a very promising therapeutic approach against cancer that is particularly effective when combined with gene therapy. Immuno-gene therapy approaches have led to the approval of four advanced therapy medicinal products (ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In spite of these remarkable successes, immunotherapy is still associated with severe side effects for CD19+ malignancies and is inefficient for solid tumors. Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop smart immunogene therapy-based-ATMPs, which can be controlled by the addition of innocuous drugs or agents, allowing the clinicians to manage the intensity and durability of the therapy. In the present manuscript, we will review the different inducible, versatile and externally controlled gene delivery systems that have been developed and their applications to the field of immunotherapy. We will highlight the advantages and disadvantages of each system and their potential applications in clinics.Spanish ISCIII Health Research FundEuropean Union (EU) PI12/01097 PI15/02015 PI18/00337 PI18/00330CECEyUCSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PAIDI-Bio326 PI-0014-2016Nicolas Monardes regional Ministry of Health 0006/2018Spanish Government FPU16/05467 FPU17/02268MCI DIN2018-01018

Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CART products delays treatment and precludes standardization. Allogeneic off-theshelf CAR-T cells are an alternative to simplify this complex and timeconsuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation aCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.Spanish ISCIII Health Research FundEuropean Commission PI15/02015 PI18/00337 PI21/00298Red TerAv RD21/ 0017/0004 PI18/ 00330 PI17/00672CECEyU and CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PAIDIBio326 CARTPI-0001- 201 PECART-0031-2020 PI0014-2016 PEER-0286-2019Spanish Government 00123009/SNEO-20191072 PLEC2021-008094regional Ministry of Health 0006/2018 C2-0002-2019Spanish Government FPU16/05467 FPU17/02268 FPU17/04327Junta de Andalucia PECART-00312020Consejeria de Salud y Familias PECART-0027-2020 MCI DIN2018-010180 DIN2020-01155

Physiological lentiviral vectors for the generation of improved CAR-T cells

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms.However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50%of the treated patients.MostCAR-Tcells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of theirmoderate andTCR-like expression profile. Compared with CAR-T cells generated with human elongation factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo.Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing ofAW-CAR-T cells in good manufacturing practice (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.Spanish ISCIII Health Research FundEuropean Commission PI15/02015 PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PA IDI-Bio326 CARTPI-0001-201 PECART-0031-2020 Red RANTECAR CAR-T 2019 00400200101918 PLEC2021-008094 PI-0014-2016 PEER-0286-2019Spanish Government PLEC2021-008094 00123009/SNEO-20191072Nicolas Monardes contracts from regional Ministry of Health 0006/2018 C2-0002-2019German Research Foundation (DFG) FPU16/05467 FPU17/02268 FPU17/04327 MCI DIN2018-010180Fundacion Andaluza Progreso y SaludGerman Research Foundation (DFG) PEJ-2018-001760-AJunta de Andalucia PE-0223-2018Biomedicine Programme of the University of Granada (Spain

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 30 long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivatorfree, all-one-one system capable of generating Dox-inducible ATMPs.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337 PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004 PI18/00330 PI17/00672Ministry of Health 2016000073332-TRA PI-57069 CARTPI-0001-201 PE-CART-0031-2020 PI-0014-2016 PECART-0027-2020 ProyExcel_00875 PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467 FPU17/02268 FPU17/04327 DIN2018-010180 DIN2020-011550 PEJ-2018-001760-

Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.This study was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI15/02015, PI18/00337 (F.M.) and PI18/00330 (K.B.) The CECEyU and CSyF of the Junta de Andalucía EDER/European Cohesion Fund (FSE) for Andalusia provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069, CARTPI-0001-201 and PAIDI-Bio326 (F.M.) and PI-0014-2016 (K.B). K.B. held a Nicolas Monardes regional Ministry of Health contract (0006/2018). S.S.H is funded thanks to Fundación Poco Frecuente donations. A.A.-G. and N.M.-P. are funded by Spanish Ministry of Science and Innovation (SMSI) through fellowships FPU17/04327 and FPU17/02268 respectively. I.R.H is funded by the ISCIII through a PFIS fellowship (FI19/00163). M.C.-G. is funded by (SMSI) through fellowship GJ (PEJ-2018-001760-A).Ye

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks

This study was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI12/01097, PI15/02015, PI18/00337 (F.M.) PIE16-00045 (J.A.M.), DTS19/00145 (J.A.M.), and PI18/00330 (K.B.) The CECEyU and CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069, and PAIDI-Bio326 (F.M.) and PI0014-2016 (K.B.). K.B. also held a Nicolas Monardes regional Ministry of Health contract (0006/2018). M.T.-M. and N.M.-P. are funded by the Spanish Ministry of Science and Innovation (SMSI) through fellowships FPU16/05467 and FPU17/02268, respectively. M.C.-G is funded by the SMSI through fellowship (PEJ-2018-001760-A). The Ramon y Cajal grant RYC-2015-18382 to P.J.R. founded by the Ministry of Economy and Competitiveness. L.L.-O. is supported by the University of Granada doctoral program (2017). M.C.-G., M.T.-M. and N.M.-P. are University of Granada Biomedicine PhD students.Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off -target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 30LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.Spanish ISCIII Health Research FundEuropean Commission PI12/01097 PI15/02015 PI18/00337 PIE16-00045 DTS19/00145 PI18/00330Junta de Andalucia 2016000073391-TRA 2016000073332-TRA PI-57069 PAIDI-Bio326 PI0014-2016Nicolas Monardes regional Ministry of Health 0006/2018Spanish Government FPU16/05467SMSI through fellowship PEJ-2018-001760-ASpanish Government RYC-2015-18382Ministry of Economy and CompetitivenessUniversity of Granada doctoral progra

Physiological lentiviral vectors for the generation of improved CAR-T cells.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products

DataSheet_1_Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma.pdf

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.</p