Digital-Droplet PCR for Quantification of CD19-Directed CAR T-Cells

Mika, Thomas; Maghnouj, Abdelouahid; Klein-Scory, Susanne; Ladigan‐Badura, Swetlana; Baraniskin, Alexander; Thomson, Julia; Hasenkamp, Justin; Hahn, Stephan A.; Wulf, Gerald; Schroers, Roland

doi:10.3389/fmolb.2020.00084

Cited by 28 publications

(37 citation statements)

References 20 publications

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“…Whole-body PET-CT scan 1 and 3 months later (days +30 and +90) showed a complete response (CR) with only residual lymph nodes without metabolic activation ( Figures 3C, D ) . The side effects and clinical response were in agreement with the expansion of anti-CD19 CAR T-cells in quantifications by ddPCR, as has recently been reported by our group ( Figure 4 ) ( 13 ).…”

Section: Case Descriptionsupporting

confidence: 91%

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Successful Chimeric Antigen Receptor (CAR) T-Cell Treatment in Aggressive Lymphoma Despite Coronavirus Disease 2019 (CoVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication - Case Report

et al. 2021

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In patients with compromised immune function, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (CoVID-19) impose particular challenges. Especially in hematological malignancies, including lymphoma, the demands by this novel virus disease are further enhanced during sophisticated treatments, such as chimeric antigen receptor (CAR) T-cell therapy. Here, we present the first case of a patient with refractory diffuse-large B-cell lymphoma, who underwent CAR T-cell treatment in the context of SARS-CoV-2. Irrespective of prolonged and active SARS-CoV-2 infection, T cells were successfully isolated by apheresis and processed to anti-CD19 CAR T cells (axicabtagene-ciloleucel). In light of the aggressive lymphoma course, lymphodepleting chemotherapy and CAR-T cells were administered in early recovery after oxygen-dependent CoVID-19 pneumonia. Except for moderate cytokine release, this cellular immunotherapy was well tolerated. Notably, there is no deterioration of the SARS-CoV-2 infection; however, complete lymphoma response and full clinical recovery were observed. In conclusion, CAR T-cell treatment in aggressive lymphoma in the setting of SARS-CoV-2 infection is feasible and may offer significant therapeutic activity in refractory disease.

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Section: Case Descriptionsupporting

confidence: 91%

“…In brief, genomic DNA was extracted from peripheral blood mononuclear cells at different time points after infusion of axi-cel. DdPCR was carried out to detect FMC63-28Z-1 amplicons as recently published by our group ( 13 ).…”

Section: Case Descriptionmentioning

confidence: 99%

Successful Chimeric Antigen Receptor (CAR) T-Cell Treatment in Aggressive Lymphoma Despite Coronavirus Disease 2019 (CoVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication - Case Report

et al. 2021

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“…From a technical perspective, different methods have been used to monitor PB CAR T-cells in patients, based either on PCR [ 40 , 41 , 42 ] or flow-cytometry [ 43 , 44 ]. As compared to PCR-based approaches, flow-cytometry CD4 and CD8 CAR + subpopulations can be distinguished [ 44 ].…”

Section: Discussionmentioning

confidence: 99%

Clinical and Product Features Associated with Outcome of DLBCL Patients to CD19-Targeted CAR T-Cell Therapy

Lamure

Laethem

Verbizier

et al. 2021

Cancers

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CD19-directed CAR T-cells have been remarkably successful in treating patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (t-FL). In this cohort study, we treated 60 patients with axicabtagene ciloleucel or tisagenlecleucel. Complete and partial metabolic responses (CMR/PMR) were obtained in 40% and 23% of patients, respectively. After 6.9 months of median follow-up, median progression-free survival (mPFS) and overall survival (mOS) were estimated at 3.1 and 12.3 months, respectively. Statistical analyses revealed that CMR, PFS, and OS were all significantly associated with age-adjusted international prognostic index (aaIPI, p < 0.05). T-cell subset phenotypes in the apheresis product tended to correlate with PFS. Within the final product, increased percentages of both CD4 and CD8 CAR+ effector memory cells (p = 0.02 and 0.01) were significantly associated with CMR. Furthermore, higher CMR/PMR rates were observed in patients with a higher maximal in vivo expansion of CAR T-cells (p = 0.05) and lower expression of the LAG3 and Tim3 markers of exhaustion phenotype (p = 0.01 and p = 0.04). Thus, we find that aaIPI at the time of infusion, phenotype of the CAR T product, in vivo CAR T-cell expansion, and low levels of LAG3/Tim3 are associated with the efficacy of CAR T-cell therapy in DLBCL patients.

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“…Other approaches that target T cell signaling sequences in quantitative PCR assays, real-time (qPCR) [ 16 ] or endpoint PCR (dPCR) [ 23 – 25 ] are suitable for CAR T cell quantification. Here, we designed our own ddPCR assays based on 2 experimental CARs with known sequences, which were validated and tested in murine experiments.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Digital-Droplet PCR for Quantification of CD19-Directed CAR T-Cells

Cited by 28 publications

References 20 publications

Successful Chimeric Antigen Receptor (CAR) T-Cell Treatment in Aggressive Lymphoma Despite Coronavirus Disease 2019 (CoVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication - Case Report

Successful Chimeric Antigen Receptor (CAR) T-Cell Treatment in Aggressive Lymphoma Despite Coronavirus Disease 2019 (CoVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication - Case Report

Clinical and Product Features Associated with Outcome of DLBCL Patients to CD19-Targeted CAR T-Cell Therapy

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

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