TATAA Blog, News

CAR-T safety monitoring


Immunosequencing entails the high-throughput sequencing of T-cell (TCR) and B-cell receptors (BCR), offering invaluable insights into the immune response within CAR-T therapies.

Immunosequencing in a laboratory

T-cell malignancies following CAR-T therapy

There is a potential risk of secondary cancers for all cell and gene therapies using integrating viral vectors [1]. Therefore, one requirement for the FDA approval of CAR-T therapies is a 15-year post-marketing follow-up to observe the potential development of secondary malignancies. In the approval summary reports, the FDA was especially wary of two possibilities:

1) Integrating viral vectors may change the expression of oncogenes and tumor suppressor genes by insertional mutagenesis, resulting in CAR-positive malignant T cells.

2) CAR-T cell-associated production of replication-competent lenti- or retrovirus in the body, which can cause secondary malignancies in other tissues.

The FDA has determined that the risk of T-cell malignancies, including chimeric antigen receptor CAR-positive lymphoma, applies to all currently approved BCMA-directed and CD19-directed genetically modified autologous CAR T cell immunotherapies and that patients and clinical trial participants receiving treatment with these products should be monitored life-long for new malignancies [2].

EMA also recognizes and considers secondary malignancies to be a significant potential risk. Close monitoring is already in place, and the marketing authorization holders of the approved medicines are required to regularly submit interim results from long-term safety studies [3]. The PRAC is now reviewing all available evidence and will decide on the need for any regulatory action.

The FDA and EMA have approved six CAR-T cell products targeting B cell cancers and multiple myeloma refractory to previous therapies. In all six products, patient T cells were genetically engineered by integrating lentiviral or retroviral vectors to express CARs targeting common B cell tumor markers CD19 and B Cell Maturation Antigen (BCMA). After activation and expansion ex vivo, the CAR-T cells are reinfused back into the same patient to attack malignant B cells. After clearance of the tumour cells, CAR-T cells can remain in the body for years and suppress potential re-emergence of malignant cells.

These treatments can suppress the bone marrow and lead to clonal hematopoiesis, a process in which stem cells harbor mutations that provide a growth advantage and preferentially expand. This can increase the patient’s risk of developing blood cancers [4]. Emerging evidence suggests that myelosuppression and clonal hematopoiesis can also be caused by CAR-T cell immunotherapies [5].

New CAR-T cell products currently in development for cancer treatment are likely to face requirements around Immune response profiling, oncogene and tumor suppressor gene expression monitoring, and risk of insertional oncogenesis.

The Cellecta workflow

TATAA has optimized the Cellecta workflow for high-throughput processing and conducted benchmarking of these workflows.

Immune receptor profiling is a powerful tool for characterizing adaptive immune responses to therapy and monitoring adverse events.

In immunosequencing the unique sequences of the T-cell and B-cell receptors (TCRs and BCRs), and antibody variable regions (CDR3) define the individual differences in adaptive immune responses.

The DriverMap AIR-RNA assay quantifies T-cell and B-cell receptor transcripts. Assaying expression of the immune receptors from either the CDR3 or the full-length receptor region enables highly sensitive detection of low-frequency, rare TCR and BCR clonotypes, and more comprehensive profiling when working with small samples and limited numbers of cells.

The DriverMap AIR-DNA assay amplifies receptor genes directly from genomic DNA. The AIR-DNA assay provides a more quantitative measurement of the genetic copies for each CDR3-specific clonotype which correlates to the number of cells with that clonotype in that sample. This data enables the measurement of clonal expansion in T and B cells.

Combining data from the AIR-DNA and AIR-RNA assays enables the assessment of transcriptional activation and the number of cells with a particular clonotype. The ability to differentiate these two effects provides a quantitative basis for assessing antigen-activated clonotypes.

Immune receptor profiling

The ratios of TCR and BCR chains were analyzed in RNA-extracted samples (50 ng) from patient plasma. All seven chains were detected in all samples from only 50ng of RNA. The graph is based on a median number of 10,000 clonotypes.

Digital PCR

Digital PCR (dPCR) is used to detect specific events such as mutations or the presence of the Chimeric Antigen Receptor (CAR) transgene. dPCR provides a highly sensitive and quantitative approach to detecting and measuring the target, complementing the immune sequencing approach using NGS technologies. TATAA Biocenter specializes in qPCR and dPCR assay development, optimization, qualification, and validation and is equipped with various dPCR instruments to cover all applications, including niched and highly multiplex assays.

Integration site analysis

The integration of viral vectors into the genome can disrupt normal gene function or lead to oncogenesis, making monitoring essential to assess the safety and efficacy of gene therapy. Next-generation sequencing (NGS) offers high sensitivity and resolution, enabling comprehensive mapping of integration sites across the genome. With our impressive array of instrumentation, we provide a range of NGS approaches and customized data analysis tailored to each project’s specific requirements.

Olink technology

Olink is designed for high-throughput protein biomarker detection using only a few microliters of sample volume, offering a streamlined, automated workflow.

High specificity is achieved by the attachment of two different antibodies to all proteins for detection.


High sensitivity is ensured by reading the barcodes through NGS or qPCR.

With 10 years of Olink proteomics experience, TATAA delivers fast, accurate protein biomarker data, accelerating your research and drug development.


TCR repertoire analysis – Cellecta

DriverMap™ AIR TCR-BCR Profiling Mouse RNA

Define proliferation and toxicity for each cell source

Olink Target 48 Mouse Cytokine panel


In vivo monitoring of CAR-T cells in blood, bone marrow, and other samples.

Determine long-term persistence of administered CAR-T cells in trial subjects.


Olink Target 48 Cytokine panels

Olink Target 96 Oncology panels

Olink Target 96 Oncology panels


DriverMap™ AIR TCR-BCR Profiling Kit (Human RNA)

DriverMap™ AIR TCR-BCR Profiling Kit (Human DNA)

  • Kohn DB, Sadelain M, Glorioso JC. Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer 2003; 3: 477–88.
  • FDA Investigating Serious Risk of T-cell Malignancy Following BCMA-Directed or CD19-Directed Autologous Chimeric Antigen Receptor (CAR) T cell Immunotherapies.
  • Meeting highlights from the Pharmacovigilance Risk Assessment Committee (PRAC) 8-11 January 2024
  • Park SJ, Bejar R. Clonal hematopoiesis in cancer. Exp Hematol 2020; 83: 105–12.
  • 360 CAR19 Therapy Drives Expansion of Clonal Hematopoiesis and Associated Cytopenias