Identification of dual positive CD19+/CD3+ T cells in a leukapheresis product undergoing CAR transduction: a case report

Abstract

Background: Chimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs.

Main body: We describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an αβ T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6 months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ 'artificial' T-cell population by the CD19-specific CAR+ T cells in culture.

Conclusions: We report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered αβhaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.

Trial registration: ClinicalTrials.gov NCT03241940.

Keywords: adoptive; cell engineering; chimeric antigen; hematologic neoplasms; immunotherapy; pediatrics; receptors.

Conflict of interest statement

Competing interests: RGM is a consultant for Lyell Immunopharma, Xyphos and GammaDelta Therapeutics. Other authors declare that they have no competing interests.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

CD19/22 CAR T+ manufacturing schema. CD19/22 CAR T cells were manufactured using the Miltenyi CliniMACs Prodigy, a closed-system manufacturing platform. The manufacturing process details are specified in the methods section in Additional File 1. For this patient’s CD19/22 CAR T+ cell product, the transduction efficiency was 48.12% and the vector copy number was 2, determined by qPCR-based detection on WHP post-transcriptional regulatory element. CAR, chimeric antigen receptor; PBMC, Peripheral blood mononuclear cells; WHP, Woodchuck. Hepatitis Virus

Figure 2

Phenotyping of apheresis and CD4+/CD8+ enriched samples. Patient apheresis and CD4+/CD8+ cells were analyzed by flow cytometry to phenotype cell populations pre-enrichment and postenrichment on the prodigy. The unusual CD3+CD19+ population (7.1%) and specifically the CD8+CD19+ population (6.5%) present in the apheresis (A) was enriched during the CD4/CD8 positive selection (B), resulting in increases of both populations, to 14.4% and 13.2%, respectively. The CD4+CD19+ population was minimal to begin with (0.62%) and increased during enrichment (1.52%). CAR, chimeric antigen receptor.

Figure 3

Rimiducid (AP1903) dose titration: 24-hour treatment of apheresis sample. Identity of the CD3+CD19+ population as the RivoCel α/β T cells containing the iCasp9 suicide gene was confirmed by rimiducid (AP1903) dose titration. PBMCs from the patient apheresis, following cryopreservation and thawing, were treated with placebo (excipient) versus AP1903 in a drug titration for 24 hours. Cryopreservation and thawing of this sample prior to analysis likely explains variability between the starting T-cell population in this figure as compared with figure 2, which was performed on fresh sample. A 78% decrease in the CD3+ CD19+ T-cell subset is demonstrated following AP1903. Non-specific decrease in CD3+ CD19− and CD3−CD19+ cell subsets is additionally seen, however effect is preferential towards CD3+CD19+ T cells (A). AP1903 treatment resulted in a dose-dependent killing of the CD3+ CD19+ population (B). CAR, chimeric antigen receptor.

Figure 4

Absence of CD3+ CD19+ iCasp9+ Cells in CD19/22 CAR T+ product. Phenotyping of the final CD19/22 CAR T+ product by flow cytometry showed the absence of this CD3+ CD19+ α/β T-cell population by day 7 (A). Reduction of the CD3+ CD19+ α/β T-cell population was confirmed by qPCR, detecting for the iCasp9 suicide gene (B). Copies of iCasp9 in the apheresis averaged 1276 copies/50 ng DNA, while the CAR product averaged 48 copies/50 ng DNA. Symbols indicate technical replicates. CAR, chimeric antigen receptor.