Antigen identification and high-throughput interaction mapping by reprogramming viral entry.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

Re-centauring T cell antigen discovery around CD4+ T cells.

In a recent issue of Cell, Dezfulian et al. develop a genome-scale platform to enable high-throughput identification of CD4+ T cell epitopes. This platform enables unbiased screens to discover antigens recognized by CD4+ T cells in cancer, infectious diseases, and autoimmunity.

A biomaterial platform for T cell-specific gene delivery.

Efficient T cell engineering is central to the success of CAR T cell therapy but involves multiple time-consuming manipulations, including T cell isolation, activation, and transduction. These steps add complexity and delay CAR T cell manufacturing, which takes a mean time of 4 weeks. To streamline T cell engineering, we strategically combine two critical engineering solutions - T cell-specific lentiviral vectors and macroporous scaffolds - that enable T cell activation and transduction in a simple, single step. The T cell-specific lentiviral vectors (referred to as STAT virus) target T cells through the display of an anti-CD3 antibody and the CD80 extracellular domain on their surface and provide robust T cell activation. Biocompatible macroporous scaffolds (referred to as Drydux) mediate robust transduction by providing effective interaction between naïve T cells and viral vectors. We show that when unstimulated peripheral blood mononuclear cells (PBMCs) are seeded together with STAT lentivirus on Drydux scaffolds, T cells are activated, selectively transduced, and reprogrammed in a single step. Further, we show that the Drydux platform seeded with PBMCs and STAT lentivirus generates tumor-specific functional CAR T cells. This potent combination of engineered lentivirus and biomaterial scaffold holds promise for an effective, simple, and safe avenue for in vitro and in vivo T cell engineering. STATEMENT OF SIGNIFICANCE: Manufacturing T cell therapies involves lengthy and labor-intensive steps, including T cell selection, activation, and transduction. These steps add complexity to current CAR T cell manufacturing protocols and limit widespread patient access to this revolutionary therapy. In this work, we demonstrate the combination of engineered virus and biomaterial platform that, together, enables selective T cell activation and transduction in a single step, eliminating multistep T cell engineering protocols and significantly simplifying the manufacturing process.

Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence.

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function-particularly at the molecular level-are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.

Peptide-MHC-targeted retroviruses enable in vivo expansion and gene delivery to tumor-specific T cells.

Tumor-infiltrating-lymphocyte (TIL) therapy has demonstrated that endogenous T cells can be harnessed to initiate an effective anti-tumor response. Despite clinical promise, current TIL production protocols involve weeks-long ex vivo expansions which can affect treatment efficacy. Therefore, additional tools are needed to engineer endogenous tumor-specific T cells to have increased potency while mitigating challenges of manufacturing. Here, we present a strategy for pseudotyping retroviral vectors with peptide-major histocompatibility complexes (pMHC) for antigen-specific gene delivery to CD8 T cells and examine the efficacy of these transduced cells in immunocompetent mouse models. We demonstrate that pMHC-targeted viruses are able to specifically deliver function-enhancing cargoes while simultaneously activating and expanding anti-tumor T cells. The specificity of these viral vectors enables in vivo engineering of tumor-specific T cells, circumventing ex vivo manufacturing processes and improving overall survival in B16F10-bearing mice. Altogether, we have established that pMHC-targeted viruses are efficient vectors for reprogramming and expanding tumor-specific populations of T cells directly in vivo , with the potential to substantially streamline engineered cell therapy production for a variety of applications.

An In Vitro Pull-down Assay of the E3 Ligase:PROTAC:Substrate Ternary Complex to Identify Effective PROTACs.

Assessing the specificity of PROTACs and confirming their proposed mechanism of action are critical for a robust targeted protein degradation program. Owing to their novel mechanism, new assays are needed to meet these goals. We and others have shown that a common explanation of PROTAC efficacy is the ability of the PROTAC to form a ternary complex between the E3 ubiquitin ligase and the target protein. In this chapter, we provide a simple in vitro method to quickly and inexpensively assess this property of PROTAC molecules. We provide detailed instructions for the purification of the specific E3 ubiquitin ligase VHL and then a generic protocol which can be adapted to any E3 ligase and substrate protein combination. This accessible method to study the ternary complex can strengthen any PROTAC-focused medicinal chemistry effort.

Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase.

PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model.

Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead.

Inhibiting protein function selectively is a major goal of modern drug discovery. Here, we report a previously understudied benefit of small molecule proteolysis-targeting chimeras (PROTACs) that recruit E3 ubiquitin ligases to target proteins for their ubiquitination and subsequent proteasome-mediated degradation. Using promiscuous CRBN- and VHL-recruiting PROTACs that bind >50 kinases, we show that only a subset of bound targets is degraded. The basis of this selectivity relies on protein-protein interactions between the E3 ubiquitin ligase and the target protein, as illustrated by engaged proteins that are not degraded as a result of unstable ternary complexes with PROTAC-recruited E3 ligases. In contrast, weak PROTAC:target protein affinity can be stabilized by high-affinity target:PROTAC:ligase trimer interactions, leading to efficient degradation. This study highlights design guidelines for generating potent PROTACs as well as possibilities for degrading undruggable proteins immune to traditional small-molecule inhibitors.

The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study.

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.