CAR-T cells are a new therapeutic tool to treat pre-B Acute Lymphoblastic Leukemia
(ALL): patient's T cells are transduced with a lentivirus to express chimeric antigen
receptors (CARs) against CD19 or CD22. However, persisting CAR-T cells generate sustained
pressure on leukemic cells that drive mechanisms of persistence such as the loss of
the targeted molecule. Indeed, 14% to 33% of relapses following CAR-CD19 therapy are
CD19-negative and occur with different CAR-CD19 products. Patients with CD19-negative
relapsed leukemia have very poor prognosis. Similarly, a mechanism of leukemic evasion
based on diminished CD22 site density has been recently reported after anti-CD22 CAR
therapy. To circumvent these limitations, we propose a therapy using CAR targeting
more than one antigen. Our hypothesis is that engineering of T cells with a bi-specific
CAR-CD19/CD22 construct could prevent the occurrence of immune-evasion and relapse
of pre-B ALL. We have graciously obtained the plasmid coding for CAR-CD22 from RJ.
Orentas (NIH). To create the CAR-CD19, we have cloned the ScFv sequence from the FMC63
anti-CD19 antibody. Using these ScFv regions, we have generated CAR constructions
containing the CD3ζ along with CD28 (2nd generation). The two CAR molecules (CD19/CD22) are constructed in a bicistronic fashion
separated by a T2A self-cleaving peptide. This transgene was cloned in a second-generation
lentiviral construct under the control of a SFFV promoter and packaged in VSVg pseudotyped
particles. These viral particulates were used to transduce peripheral CD3+ T cells, which were stimulated using Dynabeads. To test the specific cytotoxic activity
of T cells containing the double CD19-CD22 CAR in an experimental setting mimicking
tumour immune evasion, we have generated, by CRISPR, CD19 and/or CD22 knock-out ALL
cell lines. The effect of CAR-T cells was tested during a co-culture with the generated
ALL knock-out cell lines. We show that T cells bearing the dual CARs kill wild-type
ALL cell lines (CD19+CD22+) or single KO cells lines (either CD19+CD22− or CD19-CD22+) but not double KO CD19−CD22−, confirming that both CAR molecules are expressed at the cell surface and that our
bi-specific CAR-T cells can kill clones mimicking cancer immune-evasion at a single
target. This strategy to express two CARs could thus be harnessed as a way of limiting
immune-escape following CAR therapy.
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