A Rational Strategy for Reducing On-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors by Affinity Optimization

Abstract

Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using "light-chain exchange" technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated ∼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of ∼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD38 MM cells, but spared CD38 healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs.

T-cells “à la CAR-T(e)” – Genetically engineering T-cell response against cancer

“…As for TCRs, the choice of antigen is crucial to prevent offtarget effects, which can even be fatal [99,100]. This undesirable behavior may be mitigated for example by modulating the antibody affinity [101,102], by pairing high affinity heavy chains derived from a defined antibody to various light chains [103] or by mutating the signaling moiety [104].…”

Programming CAR-T cells to kill cancer

“…The relationship between binding affinity and efficacy is more nuanced in the context of CARs as compared with mAbs, for which higher affinity is typically desirable. For example, on the basis of observations from preclinical studies, whereas a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CAR derived from a high-affinity scFv (with a dissociation constant of 0.56 nM) resulted in increased therapeutic index when compared with a lower-affinity variant [28][29][30] , other examples have reported that engineering the scFv for lower affinity improves the discrimination among cells with varying antigen density [31][32][33] , which could be useful for enhancing the therapeutic window for antigens differentially expressed on tumour versus normal tissues. However, this approach might increase the risk of immune evasion, owing to the emergence of low-antigen-expressing tumour cells.…”

Chimeric antigen receptor T-cell therapies for multiple myeloma

“…79 The basis for this approach is that MM cells express higher levels of CD38 than most normal hematopoietic cells. 78,79 In addition to optimizing scFv affinity, Drent et al and Straathof et al have constructed anti-CD38 CAR-Ts with caspase-9-based suicide genes to eliminate CAR-Ts on demand.…”