Designed endocytosis-inducing proteins degrade targets and amplify signals.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

Empowering gene delivery with protein engineering platforms.

The repertoire of therapeutic proteins has been substantially augmented by molecular engineering approaches, which have seen remarkable advancement in recent years. In particular, advances in directed evolution technologies have empowered the development of custom-designed proteins with novel and disease-relevant functions. Whereas engineered proteins have typically been administered through systemic injection of the purified molecule, exciting progress in gene delivery affords the opportunity to elicit sustained production of the engineered proteins by targeted cells in the host organism. Combining developments at the leading edge of protein engineering and gene delivery has catapulted a new wave of molecular and cellular therapy approaches, which harbor great promise for personalized and precision medicine. This mini-review outlines currently used display platforms for protein evolution and describes recent examples of how the resulting engineered proteins have been incorporated into DNA- and cell-based therapeutic platforms, both in vitro and in vivo. Collectively, the strategies detailed herein provide a framework for synthesizing molecular engineering workflows with gene therapy systems for a breadth of applications in research and medicine.

Molecular Engineering of Interleukin-2 for Enhanced Therapeutic Activity in Autoimmune Diseases.

The interleukin-2 (IL-2) cytokine plays a crucial role in regulating immune responses and maintaining immune homeostasis. Its immunosuppressive effects have been harnessed therapeutically via administration of low cytokine doses. Low-dose IL-2 has shown promise in the treatment of various autoimmune and inflammatory diseases; however, the clinical use of IL-2 is complicated by its toxicity, its pleiotropic effects on both immunostimulatory and immunosuppressive cell subsets, and its short serum half-life, which collectively limit the therapeutic window. As a result, there remains a considerable need for IL-2-based autoimmune disease therapies that can selectively target regulatory T cells with minimal off-target binding to immune effector cells in order to prevent cytokine-mediated toxicities and optimize therapeutic efficacy. In this review, we discuss exciting advances in IL-2 engineering that are empowering the development of novel therapies to treat autoimmune conditions. We describe the structural mechanisms of IL-2 signaling, explore current applications of IL-2-based compounds as immunoregulatory interventions, and detail the progress and challenges associated with clinical adoption of IL-2 therapies. In particular, we focus on protein engineering approaches that have been employed to optimize the regulatory T-cell bias of IL-2, including structure-guided or computational design of cytokine mutants, conjugation to polyethylene glycol, and the development of IL-2 fusion proteins. We also consider future research directions for enhancing the translational potential of engineered IL-2-based therapies. Overall, this review highlights the immense potential to leverage the immunoregulatory properties of IL-2 for targeted treatment of autoimmune and inflammatory diseases.

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response.

Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg-expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses.

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern.

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern.

Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection.

Low-dose human interleukin-2 (hIL-2) treatment is used clinically to treat autoimmune disorders due to the cytokine's preferential expansion of immunosuppressive regulatory T cells (Tregs). However, off-target immune cell activation and short serum half-life limit the clinical potential of IL-2 treatment. Recent work showed that complexes comprising hIL-2 and the anti-hIL-2 antibody F5111 overcome these limitations by preferentially stimulating Tregs over immune effector cells. Although promising, therapeutic translation of this approach is complicated by the need to optimize dosing ratios and by the instability of the cytokine/antibody complex. We leverage structural insights to engineer a single-chain hIL-2/F5111 antibody fusion protein, termed F5111 immunocytokine (IC), which potently and selectively activates and expands Tregs. F5111 IC confers protection in mouse models of colitis and checkpoint inhibitor-induced diabetes mellitus. These results provide a roadmap for IC design and establish a Treg-biased immunotherapy that could be clinically translated for autoimmune disease treatment.