Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR

Cancers
Anita BadbaranBoris Fehse

Abstract

Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = "universal"). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samp...Continue Reading

References

Dec 1, 1991·Immunology and Cell Biology·H ZolaY Kurosawa
Dec 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·G GrossZ Eshhar
Aug 4, 1999·Proceedings of the National Academy of Sciences of the United States of America·B Vogelstein, K W Kinzler
Apr 2, 2004·Gene Therapy·B FehseC Baum
Jun 30, 2009·Journal of Immunotherapy·James N KochenderferSteven A Rosenberg
Aug 13, 2011·The New England Journal of Medicine·David L PorterCarl H June
Mar 27, 2013·The New England Journal of Medicine·Stephan A GruppCarl H June
Feb 17, 2017·Leukemia·D SommermeyerS R Riddell
Dec 12, 2017·The New England Journal of Medicine·Sattva S NeelapuWilliam Y Go
Feb 1, 2018·The New England Journal of Medicine·Shannon L MaudeStephan A Grupp
Jul 5, 2018·The New England Journal of Medicine·Carl H June, Michel Sadelain
Dec 7, 2018·The New England Journal of Medicine·Stephen J SchusterUNKNOWN JULIET Investigators
Sep 2, 2019·Recent Results in Cancer Research. Fortschritte Der Krebsforschung. Progrès Dans Les Recherches Sur Le Cancer·Astrid Holzinger, Hinrich Abken
Feb 15, 2020·Molecular Therapy. Methods & Clinical Development·Boris FehseFrancis A Ayuk
Mar 24, 2020·Molecular Therapy. Methods & Clinical Development·Alexander KunzMaria-Luisa Schubert
May 6, 2020·International Journal of Molecular Sciences·Nicoletta CoccaroFrancesco Albano

❮ Previous
Next ❯

Citations

May 21, 2021·Frontiers in Immunology·Ulrich BlacheUlrike Koehl
Nov 14, 2021·Molecular Diagnosis & Therapy·Susana Olmedillas-LópezDamián García-Olmo

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
flow-cytometry
flow cytometry
biopsies
Assay
PCRs
FACS

Software Mentioned

GraphPad Prism
cel
axi
QuantaSoft
tisa

Related Concepts

Related Feeds

CAR-T cells

Chimeric antigen receptor (CAR) T cells are cells that are genetically engineered to recognize and target specific proteins. The ability of these cells to recognize cancer antigens and eliminate tumor cells have transformed cancer immunotherapy approaches. Here is the latest research on CAR-T cells.

B cell Activation

B cell activation is initiated by the ligation of the B cell receptor with antigen and ultimately results in the production of protective antibodies against potentially pathogenic invaders. Here is the latest research.