Protocol for engineering binding domains to recognize ligand-bound receptors by using yeast surface display.

Yeast surface display is a versatile protein engineering technology, enabling precise control of the applied selection pressure. We present a yeast-surface-display-based protocol for the enrichment of binders specifically recognizing ligand-bound receptors. We describe steps for magnetic bead selections, random mutagenesis, and flow cytometric sorting, followed by library sequencing and detailed analysis of enriched clones. While this approach is exemplified with rcSso7d-based libraries and epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) complexes, it can also be adapted to other binder scaffolds and ligand-receptor systems. For complete details on the use and execution of this protocol, please refer to Dobersberger et al.1.

Small molecule-regulated switches to provide functional control of CAR T cells within the patient.

INTRODUCTION: CAR T cells have generated great excitement due to their remarkable clinical response rates in selected hematologic malignancies. However, these engineered immune cells are living drugs which are hard to control after administration. AREAS COVERED: We discuss small molecule-regulated switch systems which can potentially be used to control CAR T cell function within the patient, as well as the most important obstacles in the CAR T cell field, which might be overcome with those switch systems. EXPERT OPINION: There is an urgent need to develop advanced switch systems. Once available, we expect that they will open up new avenues for future CAR T cell generations.

An engineering strategy to target activated EGFR with CAR T cells.

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast, CAR T cell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers.

Solving the mystery of the FMC63-CD19 affinity.

The majority of approved CAR T cell products are based on the FMC63-scFv directed against CD19. Surprisingly, although antigen binding affinity is a major determinant for CAR function, the affinity of the benchmark FMC63-scFv has not been unambiguously determined. That is, a wide range of affinities have been reported in literature, differing by more than 100-fold. Using a range of techniques, we demonstrate that suboptimal experimental designs can cause artefacts that lead to over- or underestimation of the affinity. To minimize these artefacts, we performed SPR with strictly monomeric and correctly folded soluble CD19, yielding an FMC63-scFv affinity of 2-6 nM. Together, apart from analyzing the FMC63-scFv affinity under optimized conditions, we also provide potential explanations for the wide range of published affinities. We expect that this study will be highly valuable for interpretations of CAR affinity-function relationships, as well as for the design of future CAR T cell generations.

Affinity and Stability Analysis of Yeast Displayed Proteins.

Yeast surface display is a powerful protein engineering technology that is extensively used to improve various properties of proteins, including affinity, specificity, and stability or even to add novel functions (usually ligand binding). Apart from its robustness and versatility as an engineering tool, yeast display offers a further critical advantage: Once the selection campaign is finished, usually resulting in an oligoclonal pool, these enriched protein variants can be analyzed individually on the surface of yeast without the need for any sub-cloning, soluble expression, and purification. Here, we provide detailed protocols for determining both the affinity and the thermal stability of yeast displayed proteins. In addition, we discuss the advantages, challenges, and potential pitfalls associated with affinity and stability analysis using yeast surface display.

Engineering Strategies to Overcome the Stability-Function Trade-Off in Proteins.

In addition to its biological function, the stability of a protein is a major determinant for its applicability. Unfortunately, engineering proteins for improved functionality usually results in destabilization of the protein. This so-called stability-function trade-off can be explained by the simple fact that the generation of a novel protein function─or the improvement of an existing one─necessitates the insertion of mutations, i.e., deviations from the evolutionarily optimized wild-type sequence. In fact, it was demonstrated that gain-of-function mutations are not more destabilizing than other random mutations. The stability-function trade-off is a universal phenomenon during protein evolution that has been observed with completely different types of proteins, including enzymes, antibodies, and engineered binding scaffolds. In this review, we discuss three types of strategies that have been successfully deployed to overcome this omnipresent obstacle in protein engineering approaches: (i) using highly stable parental proteins, (ii) minimizing the extent of destabilization during functional engineering (by library optimization and/or coselection for stability and function), and (iii) repairing damaged mutants through stability engineering. The implementation of these strategies in protein engineering campaigns will facilitate the efficient generation of protein variants that are not only functional but also stable and therefore better-suited for subsequent applications.

Driving CARs with alternative navigation tools - the potential of engineered binding scaffolds.

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.

Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function.

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.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug.

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.