Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing Chimeric Antigen Receptors (CAR) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression but not those expressing clinically relevant GPC2 site densities (~5000 molecules/cell, range 1–6 ×10³). Iterative engineering of transmembrane (TM) and costimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen densities, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.

T cells engineered to express chimeric antigen receptors (CAR-T cells) have shown impressive clinical efficacy in the treatment of B cell malignancies. However, the development of CAR-T cell therapies for solid tumors is hampered by the lack of truly tumor-specific antigens and poor control over T cell activity. Here we present an avidity-controlled CAR (AvidCAR) platform with inducible and logic control functions. The key is the combination of (i) an improved CAR design which enables controlled CAR dimerization and (ii) a significant reduction of antigen-binding affinities to introduce dependence on bivalent interaction, i.e. avidity. The potential and versatility of the AvidCAR platform is exemplified by designing ON-switch CARs, which can be regulated with a clinically applied drug, and AND-gate CARs specifically recognizing combinations of two antigens. Thus, we expect that AvidCARs will be a highly valuable platform for the development of controllable CAR therapies with improved tumor specificity.

Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

Chimeric antigen receptors (CARs) can redirect T cells to target abnormal cells, but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a > 100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting cells (APCs) but nearly identical sensitivity when antigen is presented as purified protein. We next systematically measured the impact of engaging important T cell accessory receptors (CD2, LFA-1, CD28, CD27, and 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved the antigen sensitivity of the TCR by 125- and 22-fold, respectively, but improved CAR sensitivity by only < 5-fold. This differential effect of CD2 and LFA-1 engagement on the TCR vs. CAR was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3 ε subunit (also known as a TRuC) and fully restored by exchanging the TCR α β variable domains for those of the CAR (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors and suggest approaches to increase sensitivity.

T cells that are genetically engineered to express chimeric antigen receptors (CAR T cells) have shown impressive clinical efficacy against B‐cell malignancies. In contrast to these highly potent CD19‐targeting CAR T cells, many of those directed against other tumor entities and antigens currently suffer from several limitations. For example, it has been demonstrated that many scFvs used as antigen‐binding domains in CARs show some degree of oligomerization, which leads to tonic signaling, T cell exhaustion, and poor performance in vivo. Therefore, in many cases alternatives to scFvs would be beneficial. Fortunately, due to the development of powerful protein engineering technologies, also non‐immunoglobulin‐based scaffolds can be engineered to specifically recognize antigens, thus eliminating the historical dependence on antibody‐based binding domains. Here, we discuss the advantages and disadvantages of such engineered binding scaffolds, in particular with respect to their application in CARs. We review recent studies, collectively showing that there is no functional or biochemical aspect that necessitates the use of scFvs in CARs. Instead, antigen recognition can also be mediated efficiently by engineered binding scaffolds, as well as natural ligands or receptors fused to the CAR backbone. Finally, we critically discuss the risk of immunogenicity and show that the extent of nonhuman amino acid stretches in engineered scaffolds—even in those based on nonhuman proteins—is more similar to humanized scFvs than might be anticipated. Together, we expect that engineered binding scaffolds and natural ligands and receptors will be increasingly used for the design of CAR T cells.

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.