Rational design of chimeric antigen receptors (CARs) with optimized anti-cancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen-engagement triggers activation.We analyzed CAR-mediated antigen recognition via quantitative single molecule live-cell imaging and found the sensitivity of CAR-T-cells towards antigen approximately 1000-times reduced when compared to T-cell antigen receptor (TCR)-mediated recognition of nominal peptide/MHC complexes. While CARs outperformed TCRs with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-kinase ZAP70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR-designs, which limit at present the efficacy of CAR-T-cell therapies to target tumors with diminished antigen expression.

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 CART 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 ONswitch 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.

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.

An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.

The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is an attractive scaffold for the design of novel therapeutics. Upon engineering the C-terminal loops in the CH3 domains, Fcabs (Fc domain with antigen-binding sites) can be designed. We present the first crystal structures of Fcabs, i.e., of the HER2-binding clone H10-03-6 having the AB and EF loop engineered and the stabilized version STAB19 derived by directed evolution. Comparison with the crystal structure of the Fc wild-type protein reveals conservation of the overall domain structures but significant differences in EF-loop conformations. Furthermore, we present the first Fcab-antigen complex structures demonstrating the interaction between the engineered Fcab loops with domain IV of HER2. The crystal structures of the STAB19-HER2 and H10-03-6-HER2 complexes together with analyses by isothermal titration calorimetry, SEC-MALS, and fluorescence correlation spectroscopy show that one homodimeric Fcab binds two HER2 molecules following a negative cooperative binding behavior.