Adapter CAR T cells to counteract T-cell exhaustion and enable flexible targeting in AML

Abstract

Although the landscape for treating acute myeloid leukemia (AML) patients has changed substantially in recent years, the majority of patients will eventually relapse and succumb to their disease. Allogeneic stem cell transplantation provides the best anti-AML treatment strategy, but is only suitable in a minority of patients. In contrast to B-cell neoplasias, chimeric antigen receptor (CAR) T-cell therapy in AML has encountered challenges in target antigen heterogeneity, safety, and T-cell dysfunction. We established a Fab-based adapter CAR (AdCAR) T-cell platform with flexibility of targeting and control of AdCAR T-cell activation. Utilizing AML cell lines and a long-term culture assay for primary AML cells, we were able to demonstrate AML-specific cytotoxicity using anti-CD33, anti-CD123, and anti-CLL1 adapter molecules in vitro and in vivo. Notably, we show for the first time the feasibility of sequential application of adapter molecules of different specificity in primary AML co-cultures. Importantly, using the AML platform, we were able to demonstrate that chronic T-cell stimulation and exhaustion can be counteracted through introduction of treatment-free intervals. As T-cell exhaustion and target antigen heterogeneity are well-known causes of resistance, the AdCAR platform might offer effective strategies to ameliorate these limitations.

Chimeric antigen receptor-based natural killer cell immunotherapy in cancer: from bench to bedside

“…Shared antigenic expression of CD123 and CD33 can result in on-target nontumor toxicity [ 128 , 129 ]. In several cases, targeting various AML-associated antigens (such as FLT3 [ 130 ], CD123 [ 131 ], CD33 [ 132 ], CLL-1 [ 133 ], and GRP78 [ 134 ]) using multiple CARs may be necessary because certain tumor-associated antigens (TAAs) may not be expressed on all leukemia cells.

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Next‐generation chimeric antigen receptors for T‐ and natural killer‐cell therapies against cancer

“…An antigen‐recognition module that binds to the leucine zipper can then activate zipCAR expressing T cells upon target engagement 143 . Other novel receptor design platforms that combine a CAR backbone with a versatile extracellular domain that binds a chemical or a genetic tag linked to a tumor‐specific scFv include peptide neoepitope (PNE)‐targeting CARs, 144–146 anti‐Tag CARs (e.g., fluorescein isothiocyanate CAR), 147 SpyCatcher CAR, 148 fusion protein CAR, 149 and Fab‐based adaptor CAR (AdCAR) 150 . Bispecific antibody‐binding adaptor CARs 151 and “split CARs” that require recognition of both targeted TAAs for full activation (Figure 2H) 152–154 are other dual‐targeting approaches shown to increase tumor specificity.…”

“…143 Other novel receptor design platforms that combine a CAR backbone with a versatile extracellular domain that binds a chemical or a genetic tag linked to a tumor-specific scFv include peptide neoepitope (PNE)-targeting CARs, [144][145][146] anti-Tag CARs (e.g., fluorescein isothiocyanate CAR), 147 SpyCatcher CAR, 148 fusion protein CAR, 149 and Fab-based adaptor CAR (AdCAR). 150 Bispecific antibody-binding adaptor CARs 151 and "split CARs" that require recognition of both targeted TAAs for full activation (Figure 2H) [152][153][154] and activation after drug administration, etc. These designs have been evaluated in mouse models and some are also currently being assessed in clinical trials (Tables 1 and 2).…”

Broadening the horizon: potential applications of CAR-T cells beyond current indications

“…have recently investigated the approach of using the AdCAR platform to specifically target AML cells using three adapter molecules targeting different antigens (CD33, CD123 and CLL-1). In these ex vivo experiments, AdCAR-T cells could be applied, targeted at one antigen, and later on re-targeted towards another, achieving high anti-tumor activity ( 183 ). This approach could address the problem of therapy-induced escape variants and target downregulation on AML cells.…”