Preclinical proof of principle for orally delivered Th17 antagonist miniproteins.

Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.

Insights into cytokine-receptor interactions from cytokine engineering.

Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells.

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.

Engineering the IL-4/IL-13 axis for targeted immune modulation.

The structurally and functionally related interleukin-4 (IL-4) and IL-13 cytokines play pivotal roles in shaping immune activity. The IL-4/IL-13 axis is best known for its critical role in T helper 2 (Th2) cell-mediated Type 2 inflammation, which protects the host from large multicellular pathogens, such as parasitic helminth worms, and regulates immune responses to allergens. In addition, IL-4 and IL-13 stimulate a wide range of innate and adaptive immune cells, as well as non-hematopoietic cells, to coordinate various functions, including immune regulation, antibody production, and fibrosis. Due to its importance for a broad spectrum of physiological activities, the IL-4/IL-13 network has been targeted through a variety of molecular engineering and synthetic biology approaches to modulate immune behavior and develop novel therapeutics. Here, we review ongoing efforts to manipulate the IL-4/IL-13 axis, including cytokine engineering strategies, formulation of fusion proteins, antagonist development, cell engineering approaches, and biosensor design. We discuss how these strategies have been employed to dissect IL-4 and IL-13 pathways, as well as to discover new immunotherapies targeting allergy, autoimmune diseases, and cancer. Looking ahead, emerging bioengineering tools promise to continue advancing fundamental understanding of IL-4/IL-13 biology and enabling researchers to exploit these insights to develop effective interventions.

Engineering Anticytokine Antibodies for Immune Modulation.

The delicate balance of immune homeostasis is regulated by the interactions between cytokines and their cognate cell surface signaling receptors. There is intensive interest in harnessing cytokines as drugs for diseases such as cancer and autoimmune disorders. However, the multifarious and often contradictory activities of cytokines, coupled with their short serum half-lives, limit clinical performance and result in dangerous toxicities. There is thus growing emphasis on manipulating natural cytokines to enhance their selectivity, safety, and durability through various strategies. One strategy that has gained traction in recent years is the development of anticytokine Abs that not only extend the circulation half-life of cytokines but also specifically bias their immune activities through multilayered molecular mechanisms. Although Abs are notorious for their antagonistic activities, this review focuses on anticytokine Abs that selectively agonize the activity of the target protein. This approach has potential to help realize the clinical promise of cytokine-based therapies.

Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds.

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

De novo design of potent and selective mimics of IL-2 and IL-15.

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor ßγc heterodimer (IL-2Rßγc) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rßγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rßγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach.

Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.

Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms.

Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-ß (IL-2Rß):IL-2Rγ heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2Rα (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2Rß and IL-2:IL-2Rγ interactions, but also allosterically lowered the IL-2:IL-2Rα affinity through a "triggered exchange" mechanism favoring IL-2Rα(hi) Treg cells, creating a positive feedback loop for IL-2Rα(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rß interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rß(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.

Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps.

Interleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2Rß and γc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 "superkine" with enhanced affinity for IL-2Rß. Here, we describe next-generation IL-2 variants that function as "receptor signaling clamps." They retained high affinity for IL-2Rß, inhibiting binding of endogenous IL-2, but their interaction with γc was weakened, attenuating IL-2Rß-γc heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2Rα or IL-2Rß. Furthermore, this mutein prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Rapamycin nanoparticles increase the therapeutic window of engineered interleukin-2 and drive expansion of antigen-specific regulatory T cells for protection against autoimmune disease.

Interleukin-2 (IL-2) therapies targeting the high affinity IL-2 receptor expressed on regulatory T cells (Tregs) have shown promising therapeutic benefit in autoimmune diseases through nonselective expansion of pre-existing Treg populations, but are potentially limited by the inability to induce antigen-specific Tregs, as well as by dose-limiting activation of effector immune cells in settings of inflammation. We recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, which induce selective immune tolerance to co-administered antigens but do not increase total Treg numbers. Here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Tregs when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. Importantly, ImmTOR also increases the therapeutic window of engineered IL-2 molecules by mitigating effector immune cell expansion and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, IL-2 mutein shows potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases.

Engineered bispecific antibodies targeting the interleukin-6 and -8 receptors potently inhibit cancer cell migration and tumor metastasis.

Simultaneous inhibition of interleukin-6 (IL-6) and interleukin-8 (IL-8) signaling diminishes cancer cell migration, and combination therapy has recently been shown to synergistically reduce metastatic burden in a preclinical model of triple-negative breast cancer. Here, we have engineered two novel bispecific antibodies that target the IL-6 and IL-8 receptors to concurrently block the signaling activity of both ligands. We demonstrate that a first-in-class bispecific antibody design has promising therapeutic potential, with enhanced selectivity and potency compared with monoclonal antibody and small-molecule drug combinations in both cellular and animal models of metastatic triple-negative breast cancer. Mechanistic characterization revealed that our engineered bispecific antibodies have no impact on cell viability, but profoundly reduce the migratory potential of cancer cells; hence they constitute a true anti-metastatic treatment. Moreover, we demonstrate that our antibodies can be readily combined with standard-of-care anti-proliferative drugs to develop effective anti-cancer regimens. Collectively, our work establishes an innovative metastasis-focused direction for cancer drug development.

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.

Antibody-Invertase Fusion Protein Enables Quantitative Detection of SARS-CoV-2 Antibodies Using Widely Available Glucometers.

Rapid diagnostics that can accurately inform patients of disease risk and protection are critical to mitigating the spread of the current COVID-19 pandemic and future infectious disease outbreaks. To be effective, such diagnostics must rely on simple, cost-effective, and widely available equipment and should be compatible with existing telehealth infrastructure to facilitate data access and remote care. Commercial glucometers are an established detection technology that can overcome the cost, time, and trained personnel requirements of current benchtop-based antibody serology assays when paired with reporter molecules that catalyze glucose conversion. To this end, we developed an enzymatic reporter that, when bound to disease-specific patient antibodies, produces glucose in proportion to the level of antibodies present in the patient sample. Although a straightforward concept, the coupling of enzymatic reporters to secondary antibodies or antigens often results in low yields, indeterminant stoichiometry, reduced target binding, and poor catalytic efficiency. Our enzymatic reporter is a novel fusion protein that comprises an antihuman immunoglobulin G (IgG) antibody genetically fused to two invertase molecules. The resulting fusion protein retains the binding affinity and catalytic activity of the constituent proteins and serves as an accurate reporter for immunoassays. Using this fusion, we demonstrate quantitative glucometer-based measurement of anti-SARS-CoV-2 spike protein antibodies in blinded clinical sample training sets. Our results demonstrate the ability to detect SARS-CoV-2-specific IgGs in patient serum with precise agreement to benchmark commercial immunoassays. Because our fusion protein binds all human IgG isotypes, it represents a versatile tool for detection of disease-specific antibodies in a broad range of biomedical applications.

Weaponizing T-cell receptors through molecular engineering.

T-cell receptors (TCRs) recognize pathogens to ignite immune responses, making them attractive scaffolds for development as immunotherapeutics. However, manipulation of TCRs has been impeded by difficulties in their engineering and expression. Wagner and colleagues now establish new platforms to generate high-affinity TCR variants that potently activate T cells, and they also create soluble TCR fusion proteins that specifically recognize cognate peptides. This work provides specific tools to combat cytomegalovirus (CMV) infection and helps illuminate a general path to actuation of engineered TCR-based therapeutics.

A strategy for the selection of monovalent antibodies that span protein dimer interfaces.

Ligand-induced dimerization is the predominant mechanism through which secreted proteins activate cell surface receptors to transmit essential biological signals. Cytokines are a large class of soluble proteins that dimerize transmembrane receptors into precise signaling topologies, but there is a need for alternative, engineerable ligand scaffolds that specifically recognize and stabilize these protein interactions. Recombinant antibodies can potentially serve as robust and versatile platforms for cytokine complex stabilization, and their specificity allows for tunable modulation of dimerization equilibrium. Here, we devised an evolutionary strategy to isolate monovalent antibody fragments that bridge together two different receptor subunits in a cytokine-receptor complex, precisely as the receptors are disposed in their natural signaling orientations. To do this, we screened a naive antibody library against a stabilized ligand-receptor ternary complex that acted as a "molecular cast" of the natural receptor dimer conformation. Our selections elicited "stapler" single-chain variable fragments (scFvs) of antibodies that specifically engage the interleukin-4 receptor heterodimer. The 3.1 Å resolution crystal structure of one such stapler revealed that, as intended, this scFv recognizes a composite epitope between the two receptors as they are positioned in the complex. Extending our approach, we evolved a stapler scFv that specifically binds to and stabilizes the interface between the interleukin-2 cytokine and one of its receptor subunits, leading to a 15-fold enhancement in interaction affinity. This demonstration that scFvs can be selected to recognize epitopes that span protein interfaces presents new opportunities to engineer structurally defined antibodies for a broad range of research and therapeutic applications.

Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy.

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rß, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.