Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia

Abstract

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Immunobiology of chimeric antigen receptor T cells and novel designs

“…These “affinity‐tuned CARs” demonstrated minimal reactivity with healthy cells, while maintaining robust antitumor activity in murine models . Similarly promising results have been seen with CARs targeting ICAM‐1, CD38, and CD123 with micromolar‐range affinity, each of which exhibited improved ability to selectively kill antigen‐high target cells when compared to higher‐affinity counterparts which responded to antigen‐low targets as well …”

“…CAR T cells targeting the AML‐associated antigen folate receptor beta also demonstrated significantly improved in vitro and in vivo efficacy after a 20‐fold increase the affinity of the scFv . This principle has also been seen in TCRs targeting low site density pMHC; where increased TCR affinity allowed for enhanced responses against low antigen as long as affinity was low enough to allow for fast off‐rates and serial triggering, In contrast, a study of multiple affinity‐modified variants of a CD123 CAR demonstrated that neither increasing or decreasing the affinity of the CAR significantly altered the killing of target cells . Similarly, T cells engineered to express a high‐affinity variant of the CD22 CAR did not demonstrate improved efficacy against B‐ALL with low CD22 expression when compared to the standard affinity CD22 CAR .…”

Programming CAR-T cells to kill cancer

“…In the context of CD22-CARs, a comparison between two scFvs targeting the same epitope but with varying affinities did not reveal a significant impact of affinity on CAR function, whereas an alternative CD22 binder (m971) with lower affinity proved most efficacious, possibly because of better accessibility of the targeted epitope 34,35 . Thus, for a given antigen density on a target cell, there is likely to be an optimal range of scFv affinities and CAR expression levels required for a specific and effective antitumor response [36][37][38] .…”

“…Although targeting TAAs also expressed on normal tissues risks on-target, off-tumour toxicity, evidence from preclinical models suggests that CAR-T cells may not react to low-level antigen expression in healthy tissues 33,[36][37][38] . Instances of human epidermal growth factor receptor 2 (HER2)-CAR off-tumour toxicity 82 have been largely overshadowed by safe trials with a distinct HER2-CAR that demonstrated clinical efficacy in patients with sarcomas 91,92 .…”

Redirecting T cells with Chimeric Antigen Receptor (CAR) for the treatment of childhood acute lymphoblastic leukemia

“…Preclinical data with T cells engineered with dual-signaling receptors demonstrated that the full T-cell activation is achieved in the presence of both antigens [88]. In this context, it is possible also to tune the scFv affinity of the CAR in order to balance the efficacy with the safety [89]. Notably, dual-signaling CAR or TanCAR showed enhanced anti-tumor activity as compared to the single CAR or pooling single CARs when both antigens were expressed by the tumor [79,90], highlighting both the efficacy and the safety concerns.…”