Contributions
Abstract: S135
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 11:45 - 12:00
Location: Room A8
Background
Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expanded on those finding by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with Sleeping Beauty (SB) CD19CAR (CARCIK-CD19).
Aims
Since both non-clinical pharmacology studies in patient-derived xenograft (PDX) and toxicology of SB-transfected T cells are lacking, here we have performed a pre-clinical evaluation of donor-derived CARCIK-CD19, including validation of the GMP-compliant protocol, efficacy and biodistribution studies.
Methods
PBMC non-viral modification and CIK cell differentiation were performed according to the method enclosed in the filed patent EP20140192371 with electro-transfer of the SB GMP-grade DNA plasmids using 4D-Nucleofector (Lonza). The manufacturing process was performed in an academic Cell Factory, authorized by Agenzia Italiana del Farmaco (AIFA). Integration site (IS) analysis was performed by Sonication Linker Mediated (SLiM)-PCR and Illumina MiSeq sequencing. Human leukemia xenografts were generated in NOD.Cg-Prkdcscid Il2rgtm1Wj1/SzJ (NSG) mice. The good laboratory practice (GLP) toxicity study was conducted at GLP SR-TIGET (Milan) in absence of tumor.
Results
In a pre-GMP large-scale 18-day manufacturing process, we achieved stable CD19CAR expression (62.425%±6.399, n=8), efficient T-cell expansion (23.36±3.00-fold) and features of in vitro T-cell potency. Three lots was GMP manufactured by seeding 46.0x10^6, 56.8x10^6 and 140.4x10^6 PBMCs respectively. After 21-22 days of culture, we harvested 15.998x10^9, 1.436x10^9, and 3.897x10^9 total nucleated cells, with a mean viability of 96.96%. The median expression of CD3+CD19CAR+ cells was 46.90%, median vector copy number (VCN) was 2.0 VCN/cells and average killing was 75.29 (E:T ratio 5:1). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by IS analysis. Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in a established PDX of MLL–ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. CARCIK-CD19 induced long-term tumor regression along with a statistically significant survival prolongation compared to untreated mice (n=11, p=0.0495, Log-rank Test). Interestingly, CARCIK-CD19 cells persisted in PB and organs up to 3 months. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a bio-distribution and toxicity model. In the engrafted animals, transfected cells persisted, mainly with a constant VCN value, in the hematopoietic and post-injection perfused organs untill the end of the study (60 days) and consisted of CD8+, CD56+ and CAR+ T cells.
Conclusion
Overall, our findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing (NCT03389035).
Session topic: 25. Gene therapy, cellular immunotherapy and vaccination – Biology & Translational Research
Keyword(s): Acute lymphoblastic leukemia, Adoptive immunotherapy, Gene therapy
Abstract: S135
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 11:45 - 12:00
Location: Room A8
Background
Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expanded on those finding by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with Sleeping Beauty (SB) CD19CAR (CARCIK-CD19).
Aims
Since both non-clinical pharmacology studies in patient-derived xenograft (PDX) and toxicology of SB-transfected T cells are lacking, here we have performed a pre-clinical evaluation of donor-derived CARCIK-CD19, including validation of the GMP-compliant protocol, efficacy and biodistribution studies.
Methods
PBMC non-viral modification and CIK cell differentiation were performed according to the method enclosed in the filed patent EP20140192371 with electro-transfer of the SB GMP-grade DNA plasmids using 4D-Nucleofector (Lonza). The manufacturing process was performed in an academic Cell Factory, authorized by Agenzia Italiana del Farmaco (AIFA). Integration site (IS) analysis was performed by Sonication Linker Mediated (SLiM)-PCR and Illumina MiSeq sequencing. Human leukemia xenografts were generated in NOD.Cg-Prkdcscid Il2rgtm1Wj1/SzJ (NSG) mice. The good laboratory practice (GLP) toxicity study was conducted at GLP SR-TIGET (Milan) in absence of tumor.
Results
In a pre-GMP large-scale 18-day manufacturing process, we achieved stable CD19CAR expression (62.425%±6.399, n=8), efficient T-cell expansion (23.36±3.00-fold) and features of in vitro T-cell potency. Three lots was GMP manufactured by seeding 46.0x10^6, 56.8x10^6 and 140.4x10^6 PBMCs respectively. After 21-22 days of culture, we harvested 15.998x10^9, 1.436x10^9, and 3.897x10^9 total nucleated cells, with a mean viability of 96.96%. The median expression of CD3+CD19CAR+ cells was 46.90%, median vector copy number (VCN) was 2.0 VCN/cells and average killing was 75.29 (E:T ratio 5:1). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by IS analysis. Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in a established PDX of MLL–ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. CARCIK-CD19 induced long-term tumor regression along with a statistically significant survival prolongation compared to untreated mice (n=11, p=0.0495, Log-rank Test). Interestingly, CARCIK-CD19 cells persisted in PB and organs up to 3 months. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a bio-distribution and toxicity model. In the engrafted animals, transfected cells persisted, mainly with a constant VCN value, in the hematopoietic and post-injection perfused organs untill the end of the study (60 days) and consisted of CD8+, CD56+ and CAR+ T cells.
Conclusion
Overall, our findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing (NCT03389035).
Session topic: 25. Gene therapy, cellular immunotherapy and vaccination – Biology & Translational Research
Keyword(s): Acute lymphoblastic leukemia, Adoptive immunotherapy, Gene therapy