Impact of cryopreservation on CAR T production and clinical response

Karin Brezinger-Dayan, Orit Itzhaki*, Jenny Melnichenko, Adva Kubi, Li At Zeltzer, Elad Jacoby, Abraham Avigdor, Ronnie Shapira Frommer, Michal J. Besser*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Adoptive cell therapy with chimeric antigen receptor (CAR) T cells has become an efficient treatment option for patients with hematological malignancies. FDA approved CAR T products are manufactured in centralized facilities from fresh or frozen leukapheresis and the cryopreserved CAR T infusion product is shipped back to the patient. An increasing number of clinical centers produce CAR T cells on-site, which enables the use of fresh and cryopreserved PBMCs and CAR T cells. Here we determined the effect of cryopreservation on PBMCs and CD19 CAR T cells in a cohort of 118 patients treated with fresh CAR T cells and in several patients head-to-head. Cryopreserved PBMCs, obtained from leukapheresis products, contained less erythrocytes and T cells, but were sufficient to produce CAR T cells for therapy. There was no correlation between the recovery of PBMCs and the transduction efficacy, the number of CAR T cells obtained by the end of the manufacturing process, the in vitro reactivity, or the response rate to CAR T therapy. We could show that CAR T cells cryopreserved during the manufacturing process, stored and resumed expansion at a later time point, yielded sufficient cell numbers for treatment and led to complete remissions. Phenotype analysis including T cell subtypes, chemokine receptor and co-inhibitory/stimulatory molecules, revealed that fresh CAR T cells expressed significantly more TIM-3 and contained less effector T cells in comparison to their frozen counterparts. In addition, fresh CAR T infusion products demonstrated increased in vitro anti-tumor reactivity, however cryopreserved CAR T cells still showed high anti-tumor potency and specificity. The recovery of cryopreserved CAR T cells was similar in responding and non-responding patients. Although fresh CAR T infusion products exhibit higher anti-tumor reactivity, the use of frozen PBMCs as staring material and frozen CAR T infusion products seems a viable option, as frozen products still exhibit high in vitro potency and cryopreservation did not seem to affect the clinical outcome.

Original languageEnglish
Article number1024362
JournalFrontiers in Oncology
StatePublished - 6 Oct 2022


  • CAR T cells
  • CAR T manufacturing
  • PBMC
  • clinical trial
  • cryopreservation


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