Abstract 1541: Directed, unbiased mapping of lentiviral integrations with next generation sequencing in CAR-T cells

2018 ◽  
Author(s):  
Timothy G. Johnstone ◽  
Rajagopal Chari ◽  
David Koppstein ◽  
Ronald J. Hause ◽  
Rafael Ponce ◽  
...  
Keyword(s):  
T Cells ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Car T Cells ◽  
Car T ◽  
Generation Sequencing

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3931-3931
Author(s):  
Martina Fontaine ◽  
Benjamin Demoulin ◽  
Simon Bornschein ◽  
Susanna Raitano ◽  
Steve Lenger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Nkg2d Receptor

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

scholarly journals Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Marilia Rita Pinzone ◽  
Maria Paola Bertuccio ◽  
D. Jake VanBelzen ◽  
Ryan Zurakowski ◽  
Una O’Doherty
Keyword(s):  
T Cells ◽  
Next Generation Sequencing ◽  
Cd4 T Cells ◽  
Next Generation ◽  
Activated T Cells ◽  
Selection Pressures ◽  
Hiv Dna ◽  
Generation Sequencing

ABSTRACT Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo. When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

scholarly journals Predictive Value of Next-Generation Sequencing-Based Minimal Residual Disease after CAR-T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2842-2842
Author(s):  
Yue Huang ◽  
Houli Zhao ◽  
Mi Shao ◽  
Linghui Zhou ◽  
Guoqing Wei ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
T Cell ◽  
Cell Therapy ◽  
Patient Specific ◽  
Next Generation ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Generation Sequencing

Abstract Background Minimal residual disease (MRD) is closely associated with risk stratification of hematological malignancies. Monitoring the MRD levels of patients during treatment and at time points after remission is critical for prevention of relapse. Chimeric antigen receptor T (CAR-T) cell therapy redirects genetically modified immune cells to fight against hematologic malignancies. However, given the high relapse rate after CAR-T cell therapy, MRD monitoring by traditional techniques cannot accurately quantify the disease burden, nor can they perform high-sensitivity in-depth monitoring. Further treatment options are still controversial at such a time point when the flow cytometry (FC) results show MRD < 0.01%, while tumor clones remain. Next-generation sequencing (NGS) - MRD screens out patient-specific T/B cell receptors and accurately and quantitatively monitor patient-specific tumor cells (up to 10E-6) to reveal accurate and highly sensitive MRD levels, plus provide more timely intervention criteria. Method Between June 2016 and June 2020, we retrospectively enrolled 27 patients who achieved complete remission at the first 4-week evaluation after CAR-T cell therapy. BM samples were harvested and stored before CAR-T cell infusion and 10-154 days after infusion. We evaluated 63 specimens in total 27 patients, plus tracked immunoglobulin (IG) sequencing rearrangement by next-generation sequencing (NGS) in 20 patients. Next, we classified 17 patients into high-risk (HR, NGS-MRD positive) and low-risk (LR, NGS-MRD negative) groups according to the lower limit of NGS detection (10E-6), as well as performed leukemia-free survival (LFS) and overall survival (OS) analysis. Results At least one trackable IG clonal sequence was identified in the pre-CAR-T BM specimens from 20/27 of the cases analyzed. The two diagnostic samples with low DNA quantity, in addition to five samples lacking a dominant index sequence were excluded from further analysis. We measured the MRD in 63 samples, using a threshold of 0.01%, the determination of the presence (or absence) of leukemia was concordant in 46/63 (73%) of the samples. Discordance between NGS and FC was identified in 17/63 samples (27%). Of the 20 patients for whom trackable sequences were identified, 17 with detectable clonal index sequences on +30 days were included in our subsequent analysis cohort (Figure 1). The baseline characteristics of the patients are presented in Table 1. NGS identified 9 out of the 17 patients (52.9%) whose level of MRD was > 0.01%, but MRD negative as measured by FC. This controversial group (n=9) had inferior LFS than those whose MRD was less than 0.01% by NGS (median, 56 days vs. 219 days, p = 0.037) (Figure 2A). The OS rate was comparable between the two groups (p = 0.129) (Figure 2B). Patients with positive NGS-MRD at day 30 had comparable LFS compared with those with negative NGS-MRD (p = 0.103) (Figure 3A). For subgroup analysis, we further analyzed the influence of HSCT on prognosis of HR and LR patients. Of the total 11 patients in HR group, seven non-HSCT subjects all relapsed within three months, with a median LFS of 38 days. In contrast, the remaining four HSCT patients in HR subgroup had a significantly better LFS than the non-HSCT patients (median, 495 days vs. 38 days, p = 0.003) (Figure 3B). These four patients also exhibited better OS to that of the non-HSCT group (median, 768 days vs 409 days, p = 0.006) (Figure 3C). As for the LR cohort, no significant difference was found in LFS and OS between the HSCT and non-HSCT groups (LFS: p = 0.782, OS: p = 0.782) (Median LFS and OS data not shown). All of the evidence demonstrated that NGS-MRD early after CAR-T is an efficient prognostic factor, especially for early relapse prediction. Early post-CAR-T NGS-MRD status may be recommended for HSCT timing. As such, for HR patients, whose NGS-MRD results were positive, timely HSCT may significantly improves the prognosis and therefore is highly recommended at an early point after CAR-T cell therapy. Conclusion NGS-MRD elaborately demonstrated the tumor dynamics. NGS-MRD evaluated early after CAR-T cell therapy is an efficient prognostic factor, and timely HSCT is highly recommended for NGS-MRD positive patients at an early point after CAR-T cell therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engineering CAR-T Cells for Next-Generation Cancer Therapy

Cancer Cell ◽  
2020 ◽  
Vol 38 (4) ◽  
pp. 473-488 ◽  
Author(s):  
Mihe Hong ◽  
Justin D. Clubb ◽  
Yvonne Y. Chen
Keyword(s):  
T Cells ◽  
Cancer Therapy ◽  
Next Generation ◽  
Car T Cells ◽  
Car T
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