scholarly journals Detection of Vector Copy Number in Bicistronic CD19xCD22 CAR T Cell Products with Digital PCR

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4001-4001
Author(s):  
Lindsey A. Murphy ◽  
Russell Marians ◽  
Mark Eric Kohler ◽  
Terry J. Fry ◽  
Amanda C. Winters
Keyword(s):  
T Cells ◽  
T Cell ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Cell Product ◽  
Car T Cell ◽  
Car T ◽  
Vector Copy Number ◽  
Labeled Probe

Abstract Chimeric antigen receptor (CAR) T cell therapy is a rapidly evolving immunotherapeutic treatment modality for adult and pediatric patients with a variety of cancers, which has been most extensively investigated in B-cell malignancies. Given that CAR T cell immunotherapy involves changing the genetic composition of a patient's T cells, this living drug presents unique safety and quality control challenges. Vector copy number (VCN), a measurement of transgene copies within a CAR T cell product, is a product-specific characteristic that must be quantified prior to patient administration as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T cell products has been performed via qPCR. qPCR is reliable along a broad range of concentrations but has inherent limitations in its lower limit of detection and limit of quantification. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared to qPCR include simplicity, reproducibility, lower limit of detection, and definitive quantification. In this present study, we developed an assay for analysis of the novel bicistronic UCD19x22 CAR T cell construct, which was developed in the laboratory of Dr. Terry Fry at the University of Colorado and will be moving in to clinical trials later this year. Custom primer-probe assays were designed using Primer Express v3.0.1 and the ThermoFisher Custom TaqMan Assay Design Tool. As an internal control, forward and reverse primers as well as a VIC-labeled probe specific to human albumin (NCBI gene 213, HGNC:399) were designed. Primers and a FAM-labeled probe assay, specific for the bicistronic CD19x22 CAR T cell product, were designed at the junction site between the two distinct CARs. This study compares two different digital PCR modalities: (1) droplet digital PCR (ddPCR) via the BioRad QX200 system which utilizes water-in-oil droplet partitions and (2) the QIAcuity digital PCR system utilizing a nanoplate-based partitioning platform. While dPCR is a newer methodology compared to ddPCR, the two apply parallel procedures, data generation, and analyses. The primer/probe assay was validated with qPCR, dPCR and ddPCR using patient samples from preclinical CAR T cell manufacturing production runs, as well as Jurkat cell subclones which stably express this bicistronic CAR T product. We successfully developed an assay to specifically detect and quantify our bicistronic CD19xCD22 CAR transgene. ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product without any signal detected in samples containing untransduced T cells or T cells transduced with CD19 only CARs. Additionally, our assay gives accurate, precise, and reproducible CAR T cell VCN measurements across qPCR, dPCR, and ddPCR modalities. We demonstrate that digital PCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, and that specific assays can be developed for detection of unique constructs. Future studies will evaluate the utility of this assay with digital PCR modalities in measuring CAR T cell persistence in clinical trial patient samples after receiving this novel CAR T cell product. Figure 1 Figure 1. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

scholarly journals OMIC-11. SINGLE CELL RNA SEQUENCING FROM THE CSF OF SUBJECTS WITH H3K27M+ DIPG/DMG TREATED WITH GD2 CAR T-CELLULAR THERAPY

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i39-i39
Author(s):  
Aaron Mochizuki ◽  
Sneha Ramakrishna ◽  
Zina Good ◽  
Shabnum Patel ◽  
Harshini Chinnasamy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Product ◽  
Myeloid Suppressor Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Single Cell Rna Sequencing

Abstract Introduction We are conducting a Phase I clinical trial utilizing chimeric antigen receptor (CAR) T-cells targeting GD2 (NCT04196413) for H3K27M-mutant diffuse intrinsic pontine glioma (DIPG) and spinal cord diffuse midline glioma (DMG). Cerebrospinal fluid (CSF) is collected for correlative studies at the time of routine intracranial pressure monitoring via Ommaya catheter. Here we present single cell RNA-sequencing results from the first 3 subjects. Methods Single cell RNA-sequencing was performed utilizing 10X Genomics on cells isolated from CSF at various time points before and after CAR T-cell administration and on the CAR T-cell product. Output was aligned with Cell Ranger and analyzed in R. Results As detailed in the Majzner et al. abstract presented at this meeting, three of four subjects treated at dose-level one exhibited clear radiographic and/or clinical benefit. We have to date completed single cell RNA-sequencing for three of these four subjects (two with benefit, one without). After filtering out low-quality signals and doublets, 89,604 cells across 3 subjects were analyzed. Of these, 4,122 cells represent cells isolated from CSF and 85,482 cells represent CAR T-cell product. Two subjects who demonstrated clear clinical and radiographic improvement exhibited fewer S100A8+S100A9+ myeloid suppressor-cells and CD25+FOXP3+ regulatory T-cells in the CSF pre-infusion compared to the subject who did not derive a therapeutic response. In one subject with DIPG who demonstrated improvement, polyclonal CAR T-cells detectable in CSF at Day +14 demonstrated enrichment of CD8A, GZMA, GNLY and PDCD1 compared to the pre-infusion CAR T-cells by trajectory analysis, suggesting differentiation toward a cytotoxic phenotype; the same subject exhibited increasing numbers of S100A8+S100A9+ myeloid cells and CX3CR1+P2RY12+ microglia over time. Further analyses will be presented as data become available. Conclusions The presence of immunosuppressive myeloid populations, detectable in CSF, may correlate to clinical response in CAR T cell therapy for DIPG/DMG.

scholarly journals Efficacy and Safety of CAR-T Therapy with Safety Switch Targeting Bcma for Patients with Relapsed/Refractory Multiple Myeloma in a Phase 1 Clinical Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3154-3154 ◽  
Author(s):  
Weijun Fu ◽  
Juan Du ◽  
Hua Jiang ◽  
Zhi CHENG ◽  
Runhong Wei ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Phase 1 ◽  
Cellular Immunotherapy ◽  
Infusion Therapy ◽  
Cell Product ◽  
Car T Cell ◽  
Car T

Background: Encouraging results are seen from several early phase clinical trials on the cellular immunotherapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) targeting B cell maturation antigen (BCMA) for the treatment of relapsed/refractory (RR) multiple myeloma (MM). We developed an anti-BCMA CAR-T cell product manufactured via gamma-retrovirus-mediated transduction of activated T cells to express a second-generation CAR with the 4-1BB costimulatory domain along with a truncated epidermal growth factor receptor (tEGFR) as a safety switch. The preclinical study confirmed its high reactivity against MM cells. Methods: A phase 1 clinical trial (NCT03093168) has been launched to evaluate the safety and feasibility of this BCMA CAR-T cell product for treating RRMM. The enrolled RRMM patients had received at least 2 prior treatment regimens, including a proteasome inhibitor and an immunomodulatory agent, or are double-refractory, and have over 5% BCMA expression on plasma cells (Nine patient with extramedullary plasmacytoma does not express BCMA). Patients were subjected to a lymphodepleting regimen with Cy (300 mg/m2, d-5 to d-3) and Flu daily for 3 days (25 mg/m2, d-5 to d-3) prior to the CAR-T infusion (d0) at a dose of 9×106CAR+ cells/kg. The efficacy was assessed by the International Uniform Response Criteria for Multiple Myeloma, and the toxicity is graded by CTCAE 4.03. Results: As of March 1th, 2019, 46 patients had been infused with this intended dose of the autologous BCMA CAR-T cells, and 44 patients had reached at least 1 month of follow-up. As of this data cut-off, the overall response rate (ORR) for the 44 evaluable patients was 79.6%, including 2sCRs, 16CRs, 8VGPRs and 8PRs, and 16 patients reached MRD-negative response. The CAR-T cell expansion and persistence were consistently observed throughout these patients. The medianPFS is 15mon, and the median OS result has not been reached (49.16% progression-free survival, and 53.95% overall survival at 24 months). Among the 44 infused patients, 22.7% had grade 1-2 Cytokine release syndrome (CRS ) and 6.8% (3 patients) had grade 3 CRS. No grade 4 CRS reactions developed and all toxicities were fully reversible. Conclusions: Our result demonstrates the high potential of this single CAR-T infusion therapy for RRMM, including 2sCRs, 16CRs and ongoing clinical responses for more than 26 months, with manageable CRS to date. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Disclosures No relevant conflicts of interest to declare.

Effect of chimeric antigen receptor (CAR) T cells on clonal expansion of endogenous non-CAR T cells in patients (pts) with advanced solid cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 3011-3011 ◽  
Author(s):  
Rebecca H Kim ◽  
Gabriela Plesa ◽  
Whitney Gladney ◽  
Irina Kulikovskaya ◽  
Bruce L Levine ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clonal Expansion ◽  
Time Points ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Vaccine Effect

3011 Background: CAR T cells have produced remarkable responses in heme malignancies, but efficacy in solid cancers is limited. Poor in vivo persistence and heterogeneous expression of the CAR target on tumors are potential barriers to the success of CAR T cell therapy. However, even with transient persistence, CAR T cells may elicit a “vaccine” effect by inducing cancer cell death and subsequent release of tumor antigens that could stimulate tumor-specific T cell activity. Methods: 6 pts with pancreatic ductal adenocarcinoma (PDAC) received repeated 3x per week intravenous (iv) infusions of mRNA-transfected mesothelin-redirected CAR T cells (CARTmeso). Pts with PDAC (n = 5), ovarian carcinoma (n = 5), and mesothelioma (n = 5) received iv infusion of lentiviral-transduced (lenti) CARTmeso with or without cyclophosphamide (Cy) preconditioning. Peripheral blood samples were collected from pts at baseline and defined time points after treatment. Genomic DNA from these samples or from pre-infused CAR T cell product was used for deep sequencing of the TCRbeta chain using the ImmunoSEQ platform. A TCRbeta clone was considered to have expanded from baseline to defined time points after treatment if it showed a two-fold change from baseline and met statistical significance by Fisher’s exact test (p < 0.05). Results: mRNA CARTmeso cells persisted in vivo for < 24 hrs. Unexpectedly, therapy induced clonal T cell expansion detected in the blood by day 14 in all 6 pts. Expanded clones underwent contraction by day 28 in 3 pts. In one pt, peripherally expanded clones were also detected in a tumor biopsy, but without significant intratumoral clonal expansion. Lenti CARTmeso therapy also induced peripheral expansion of T cell clones both present and not present in the infused CAR T cell product. However, with Cy preconditioning, clonal expansion seen after lenti CARTmeso therapy was predominately restricted to clones detected in the CAR T cell product. Conclusions: In pts with advanced solid cancers, CARTmeso stimulates clonal expansion of endogenous T cells, which is lost with Cy conditioning. Findings suggest that CAR T cells may elicit a “vaccine” effect with potential therapeutic implications.

Efficacy of SCRI-CAR19x22 T cell product in B-ALL and persistence of anti-CD22 activity.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3035-3035
Author(s):  
Rebecca Alice Gardner ◽  
Colleen Annesley ◽  
Ashley Wilson ◽  
Corinne Summers ◽  
Prabha Narayanaswamy, ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Grade 3 ◽  
Dose Levels ◽  
Negative Population

3035 Background: Loss of CD19 expression is a major cause of limited durable B-ALL remission following CD19 CAR T cells, which might be overcome by utilization of dual CD19xCD22 CAR T cell targeting. Methods: A Phase I trial (NCT03330691) of SCRI-CAR19x22 was developed using dual transduction of lentiviral vectors encoding for either a CD19- or CD22-specific CAR T cell construct, both with 4-1BB co-stimulation. Manufacturing was performed in a closed G-Rex system with IL-7, IL-15 and IL-21. After lymphodepletion, CAR T cells were infused at 1 or 3 X 106 CAR T cells/kg dose levels. Leukemic response and CAR T cell persistence were evaluated by flow cytometry. Results: Products were successfully manufactured in all 28 enrolled subjects with 7.92 average days in culture (range of 7-11 days) and consisted of an average CD8:CD4 ratio of 3.09 (range 0.19 to 8.9). The cellular product CAR composition was 29% CD19, 31% CD22 and 39% CD19 and CD22 targeting. 13 subjects had prior exposure to CD19 or CD22 targeting therapies with diverse expression of CD19 and CD22 on the leukemic blasts. No dose limiting toxicities occurred in the 27 infused subjects. The recommended phase 2 dose is 3 x 106 CAR+ cells/kg. CRS was present in 80% of subjects, with 85% of CRS being grade 2 or less, and a peak grade of 3 (n = 3). Mild neurotoxicity occurred in 38%, with a single grade 3 event. 84.6% obtained a CR, of which 95% were MRD negative. Of the 4 subjects who did not achieved a CR, 2 had a pre-existing CD19 negative population and one had previously received CAR T cells and rejected SCRI-CAR19x22. There have been 4 relapses with varying CD19 and CD22 expression as follows: 1 CD19-CD22-, 1 CD19+CD22+, and 2 CD19-CD22+. The in vivo engraftment of CAR T cells peaked most frequently between day +7 and +14 and was predominated by the CD19 CAR+ T cells. Conclusions: We demonstrate manufacturing feasibility and safety of SCRI-CAR19x22. While initial efficacy is demonstrated, CD22 activity is poor due to limited expansion of the CD22 CAR-containing components and subjects with pre-existing CD19 negative leukemia fared poorly. Development of a revised CD22 CAR that exhibits a reduction tonic signaling is underway, with plans to explore the new construct in the context of a dual-targeting CD19xCD22 CAR T cell product. Clinical trial information: NCT03330691 .

CD19 CAR T-cell product type independently impacts CRS and ICANS severity in patients with aggressive NHL.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 7532-7532
Author(s):  
Jordan Gauthier ◽  
Aisling Cearley ◽  
Paula Perkins ◽  
Angela Kirk ◽  
Mazyar Shadman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hematopoietic Cell Transplantation ◽  
Product Type ◽  
Multivariable Model ◽  
T Cell Therapy ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

7532 Background: CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T cells achieve high response rates in patients (pts) with relapsed or refractory (R/R) aggressive B-cell non-Hodgkin lymphoma (NHL), but are limited by cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Pivotal trial data suggested distinct toxicity risks across CD19 CAR T-cell products, but differences in pt and disease characteristics may have confounded these observations. Thus, we assessed the independent impact of 3 CD19 CAR T-cell products (axicabtagene ciloleucel[axicel], tisagenlecleucel [tisacel], and JCAR014) on CRS and ICANS severity in 136 pts with R/R aggressive NHL. Methods: We retrospectively analyzed aggressive NHL pts treated at our institutions with cyclophosphamide and fludarabine lymphodepletion (LD) followed by CD19 CAR T-cell therapy. Axicel and tisacel pts were treated off trial using commercial products. JCAR014 (defined-composition 4-1BB-costimulated CD19 CAR T cells) was administered in all pts at the dose of 2x106/kg on a phase I/II clinical trial (NCT01865617). CRS and ICANS were graded according to the ASTCT criteria and CTCAE 4.03, respectively. We used multivariable proportional odds logistic regression to model CRS and ICANS grade. Results: The CAR T-cell product was axicel, tisacel, or JCAR014 in 50%, 28%, and 22% of pts, respectively. Compared to axicel pts, we observed higher preLD LDH levels in tisacel and JCAR014 pts, and lower preLD albumin with tisacel (p < 0.001) with comparable age and hematopoietic cell transplantation comorbidity (HCT-CI) indexes across CAR T-cell products. Higher day-28 overall response rate by Lugano criteria was observed after axicel (71%) compared to tisacel (56%) and JCAR014 (53%). Adjusting for age, HCT-CI, preLD LDH, preLD albumin, CAR T-cell product type was associated with CRS severity (tisacel versus [vs] axicel, OR = 0.45, p = 0.05; JCAR014 vs axicel, OR = 0.29, p = 0.005;). Age had limited or no impact on CRS severity (OR 95%CI, 0.97-1.02), while the effect of HCT-CI was undetermined (OR 95%CI, 0.85-1.27). In a multivariable model including the same covariates as above, CAR T-cell product type (tisacel vs axicel, OR =.14, p <.001; JCAR014 vs axicel, OR = 0.31, p = 0.009), preLD LDH (OR, 3.96 per log10 increase; p = 0.04) and age (OR per 10-year increase, 1.32; p =.06) were associated with ICANS severity. Interaction effect testing suggested effect modification of age by the CAR T-cell product type (tisacel/JCAR014 versus axicel, p = 0.06); using a multivariable model including this interaction term, the predicted probabilities of grade ≥3 ICANS in a 70 year-old after axicel, tisacel, and JCAR014 were 40%, 6%, and 8%, respectively. Conclusions: CAR T-cell product type independently impacts CRS and ICANS severity in NHL pts. Our findings provide key insights to guide patient and CAR T-cell product selection.

scholarly journals JWCAR029 Is a CD19-Targeted CAR T Cell Product with Process and Quality Controls Delivered As a Flat Dose of CAR T Cell to Patients with NHL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5387-5387 ◽  
Author(s):  
Wen Wang ◽  
Ming Hao ◽  
Yin Cheng ◽  
Juan Gao ◽  
Su Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dose Level ◽  
Employment Equity ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Flat Dose ◽  
Car T ◽  
Equity Ownership

Abstract Background: JWCAR029 is the first IND approved CD19-targeted CAR T cell product by China National Drug Administration (CNDA) containing 4-1BB as the co-stimulatory factor with highly reproducible process and quality control that allows flat dose of CAR T cell infusion. To date, a total of 22 lots have been manufactured and 18 subjects have been infused in the ongoing multicenter, Phase 1 trial (NCT03344367 and NCT03355859) evaluating the safety and efficacy of JWCAR029 in adult relapsed or refractory B-cell Non-Hodgkin lymphoma patients. The process and quality control strategy for JWCAR029 contributes to the low variability in drug product quality attributes. Methods: Manufacturing of JWCAR029 begins with patient derived autologous T cells obtain via apheresis. JWCAR029 drug products were analyzed for viability, potency, subtype of T cells, copy numbers of lentiviral vector, and cell health related attributes using cellometer related bioassays, flow cytometry, and real-time quantitative polymerase chain reaction system (qPCR), respectively. Results: Process and quality of JWCAR029 started with an automated wash and T cell purification that results in pure CD3+ populations (median 99.56%, Inter Quartile Range [IQR] 99.22-99.86%). CD3+ T cells were transduced with lentiviral vector expressing a CD19-directed CAR with a 4-1 BB/CD3ζ endodomain. CAR+ T cells were cultured to a target cell dose and then formulated / cryopreserved for infusion. To reduce between-lot variance, the cryopreserved drug product (CDP) was packaged at fixed volume with a tight range of viable cell concentrations (CD3+: median 40.25 × 10^6 cells/mL, IQR 31.10-69.13 × 10^6 cells/mL, N=22) and CD3+CAR+ cell concentrations (median 27.25 × 10^6 cells/mL, IQR 23.57-33.10 × 10^6 cells/mL, N=22). JWCAR029 does not use a fixed ratio of CD4+CAR+ cells/CD8+CAR+ cells in the final CDP (median 1.18, IQR 0.70-1.95, N=22). In the ongoing, multicenter, single arm, open-label and dose escalation Phase 1 trial, JWCAR029 was administered as a flat dose at dose level 1 (DL1) of 2.5 × 10^7 CAR+ T cells (6 subjects), at dose level 2 (DL2) of 5.0 × 10^7 CAR+ T cells (9 subjects), or dose level 3 (DL3) of 1.0 × 10^8 CAR+ T cells (3 subjects). After infusion, stable expansion of CD4+ and CD8+ CAR+ T cells were observed and peak value was appeared at day 11 to day 15 after administration. Low occurrence rate and manageable cytokine release syndrome (CRS) and neurotoxicity (NT) with high complete response (CR) rate were observed with emerging dose: response relationship. Detailed PK, clinical safety, and efficacy data of JWCAR029 will be presented separately. Conclusion: In order to employ standardized and high quality cell therapy methods in a Chinese multi-center trial, JWCAR029 was developed to provide a CD19-directed 4-1BB CAR T cell product with highly controlled manufacturing and quality processes enables administration in adult relapsed or refractory B-cell Non-Hodgkin lymphoma subjects. These control strategies in manufacturing and quality processes facilitated to the low rates of CRS and NT. Disclosures Hao: JW Therapeutics: Employment, Equity Ownership. Cheng:JW Therapeutics: Employment, Equity Ownership. Gao:JW Therapeutics: Employment, Equity Ownership. Liu:JW Therapeutics: Employment, Equity Ownership. Lam:JW Therapeutics: Consultancy. Yao:JW Therapeutics: Employment, Equity Ownership; WuXi AppTec: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

TRANSCEND CLL 004: Minimal residual disease (MRD) negative responses after lisocabtagene maraleucel (Liso-Cel; JCAR017), a CD19-directed CAR T cell product, in patients (pts) with relapsed/refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL/SLL).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 7501-7501 ◽  
Author(s):  
Tanya Siddiqi ◽  
Kathleen Anne Dorritie ◽  
Jacob Drobnyk Soumerai ◽  
Deborah Marie Stephens ◽  
Jason A Dubovsky ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Complete Blood Count ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

7501 Background: Eradication of MRD in CLL patients may be necessary for deep and durable responses. We assessed safety, pharmacokinetics, and efficacy of liso-cel, an investigational, anti-CD19 CAR T cell product administered as a defined composition of CD4+/CD8+ CAR T cells, in the ongoing phase 1/2 TRANSCEND CLL 004 study. Methods: Eligible pts had CLL/SLL, received ≥2 prior lines of therapy (including Bruton’s tyrosine kinase inhibitors [BTKi] unless medically contraindicated), and had ECOG PS ≤1. Post lymphodepleting chemotherapy, pts received liso-cel infusion at either dose level (DL)1 = 50 × 106 or DL2 = 100 × 106 total CAR+ T cells. Patients were monitored for dose-limiting toxicities (DLTs). Response was assessed by iwCLL 2008 criteria. MRD was assessed by flow cytometry in blood (10−4) and by NGS in bone marrow (BM; 10−6). Results: At data cutoff, 16 pts received liso-cel: DL1, n = 6; DL2, n = 10. 75% of pts had high-risk features ( TP53 mutation, complex karyotype, or del17p); 100% had prior ibrutinib and 50% had prior venetoclax. Median (range) number of prior lines of therapy was 4.5 (2‒11). There was 1 DLT of grade (G) 4 hypertension (DL2). The most common G3/4 treatment-emergent adverse events were cytopenias (thrombocytopenia, 75%; anemia, 69%; neutropenia, 63%; leukopenia, 56%). 1 pt had G3 cytokine release syndrome (CRS); 3 pts had G3 neurological events (NE). Best overall response rate (ORR) in 15 evaluable pts was 87% (13/15). 7 pts (47%) achieved complete remission with/without complete blood count recovery (CR/CRi). ORR at 6 mo was 83% (5/6). 10/15 pts (67%) achieved undetectable MRD (uMRD) in blood by day 30 and in 7/8 pts (88%) in BM. MRD-negative CRs were seen in patients who had failed both BTKi and venetoclax. Median time to peak blood CAR+ T cell level was 16 days (4‒30). Conclusions: In this study of heavily pretreated pts with standard- and high-risk CLL/SLL and previous ibrutinib treatment, liso-cel-related toxicities (ie, CRS and NEs), were manageable. Pts rapidly achieved CR/CRi and uMRD. Additional follow-up will be presented. Clinical trial information: NCT03331198.

scholarly journals Droplet digital PCR allows vector copy number assessment and monitoring of experimental CAR T cells in murine xenograft models or approved CD19 CAR T cell-treated patients

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Rafik Haderbache ◽  
Walid Warda ◽  
Eric Hervouet ◽  
Mathieu Neto da Rocha ◽  
Rim Trad ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Copy Number ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Vector Copy Number ◽  
Pcr Assays

Abstract Background Genetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer. Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells. Methods Based on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs. Results 28BBz and 28z ddPCR assays applied to 2 tumoral (acute myeloid leukemia (AML) or multiple myeloma (MM) xenograft humanized NSG mouse models allowed us to quantify the early expansion (up to day 30) of CAR T cells after injection. Interestingly, following initial expansion, when circulating CAR T cells were challenged with the tumor, we noted a second expansion phase. Investigation of the bone marrow, spleen and lung showed that CAR T cells disseminated more within these tissues in mice previously injected with leukemic cell lines. Finally, circulating CAR T cell ddPCR monitoring of R/R acute lymphoid leukemia or diffuse large B cell lymphoma (n = 10 for tisa-cel and n = 7 for axi-cel) patients treated with both approved CAR T cells allowed detection of early expansion, which was highly correlated with FC, as well as long-term persistence (up to 450 days), while FC failed to detect these events. Conclusion Overall, we designed and validated 2 ddPCR assays allowing routine or preclinical monitoring of early- and long-term circulating approved or experimental CAR T cells, including our own IL-1RAP CAR T cells, which will be evaluated in an upcoming phase I clinical trial.

scholarly journals The fist experience of using locally manufactured CAR-T cells in patients with relapsed/refractory acute lymphoblastic leukemia in Belarus

2021 ◽  
Vol 20 (2) ◽  
pp. 30-38
Author(s):  
O. V. Aleinikova ◽  
A. A. Migas ◽  
E. A. Stolyarova ◽  
A. V. Punko ◽  
L. V. Movchan ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Pediatric Oncology ◽  
Lymphoblastic Leukemia ◽  
High Dose ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

The results of treatment of recurrent/refractory acute lymphoblastic leukemia (ALL) with both standard and high-dose chemotherapy are unsatisfactory and require the development of new therapeutic options. The use of immunotherapy approaches opens up new perspectives for patients whose cytotoxic chemotherapy was ineffctive or intolerable. This article describes the experience of using CD19 CAR-T cells manufactured at the Republican Scientifi and Practical Center for Pediatric Oncology, Hematology and Immunology after lymphodepletion with fldarabine and cyclophosphamide in two patients over 18 years of age with refractory relapse of ALL. Other possibilities of conservative treatment for these patients have been exhausted. The study was approved by the Independent Ethics Committee and the Scientifi Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (Republic of Belarus). The chimeric 2nd generation receptor was constructed from the anti-CD19 scFv antibody fragment, the CD28 transmembrane domain, signaling domains of the 4-1BB and CD3z proteins, and transduced into T-lymphocytes as part of the pWPXL lentiviral vector. The cell product was obtained by separation and separate processing of CD4 and CD8 lymphocytes in the presence of IL-7 and IL-15. The subpopulation composition of the resulting CAR-T cell product and the expression of immune checkpoints were assessed. The results obtained indicate a high antileukemic activity of the obtained CAR-T cells. Monitoring of CAR-T cells' persistence, the level of minimal residual disease, and the spectrum of inflmmatory cytokines in the blood was performed. Both patients responded to CAR-T therapy by lowering their blast cell levels. Treatment was accompanied by a cytokine release syndrome controlled by a recombinant monoclonal antibody to the human IL-6 receptor, tocilizumab. The developed and replicated laboratory-derived CAR-T cell technology can be used to treat patients with severe relapsed/refractory B-line ALL as rescue therapy and provide additional chances for their cure.

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