CRISPR-based gene disruption and integration of high-avidity, WT1-specific T cell receptors improve antitumor T cell function

Abstract

T cell receptor (TCR)–based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms’ tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01–restricted TCRs, three that were specific to the less explored immunodominant WT1 37–45 and two that were specific to the noncanonical WT1 −78–64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant ( TRAC ) locus with TCR β constant ( TRBC ) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT1 37–45 -specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

Non-viral precision T cell receptor replacement for personalized cell therapy

“…Precision gene-editing using techniques such as CRISPR–Cas9 enables the simultaneous knockout of endogenous TCR chains while inserting the transgenic TCR under the control of the physiological TCR promoter. This strategy has been reported in some settings 17 , 18 , but not all 19 , to provide advantages over the same transgenes expressed under the control of constitutive viral vector promoters.…”

Targeting an alternate Wilms’ tumor antigen 1 peptide bypasses immunoproteasome dependency

“…5A) ( 30 ). Our screen identified WT1 37–45 [also identified by Ruggiero et al ( 31 )] and WT1 235–243 as promising immunogenic candidates. Cell lines targeting WT1 37–45 and WT1 235–243 were sorted on tetramer positivity (Fig.…”

“…We pursued the latter strategy in searching for an epitope not exclusively dependent on the immunoproteasome. Our results suggest that selecting T cells/TCRs recognizing WT1 37–45 [see also Ruggiero et al ( 31 )] rather than WT1 126–134 should assure that changes in the relative immunoproteasome expression cannot abrogate presentation of WT1-expressing cells and allow for immune evasion, thus promoting successful clinical translation for both vaccines and TCR-engineered T cell therapies.…”

Human vaccines & immunotherapeutics: news February 2022

“…The treatment also overcame resistance in one patient, who had relapsed following earlier TCR therapy targeted against a different region of the WT1 protein. 7 , 8 …”