Immune checkpoint blockade (ICB) has significantly improved the survival for many patients with advanced malignancy. However, fewer than 50% of patients benefit from ICB, highlighting the need for more effective immunotherapy options. High-dose interleukin-2 (HD IL-2) immunotherapy, which is approved for patients with metastatic melanoma and renal cell carcinoma, stimulates CD8+ T cells and NK cells and can generate durable responses in a subset of patients. Moreover, HD IL-2 may have potential efficacy in patients whose disease has progressed following ICB and plays a vital role in expanding tumor-infiltrating lymphocyte (TIL) in TIL therapy. Despite its potential, the use of HD IL-2 is limited by severe toxicities such as hypotension and vascular leak syndrome. Additionally, only a few patients achieve a good outcome after HD IL-2 therapy. To address these challenges, numerous next-generation IL-2 receptor (IL-2 R) agonists have been developed to exhibit treatment effects while minimizing adverse events. This review will explore IL-2 biology, the clinical application of HD IL-2 therapy, and the development of novel IL-2 R agonists for cancer immunotherapy.

As T and NK cell exhaustion is attributed to increased expression of immune checkpoints and decreased production of proliferative cytokines by these cells, immune checkpoint-targeted delivery of proliferative cytokines might induce robust and sustained antitumor immune responses. Here, the expression profile of NKG2A was first found to be narrower than that of PD-1 in tumor-infiltrated immune cells. Moreover, unlike PD-1, NKG2A was predominantly co-expressed with IL-2Rβγ in tumor-infiltrated CD8+ T and NK cells, but not in Tregs, suggesting that NKG2A might be an ideal target for delivery of IL-2Rβγ agonists to overcome T and NK exhausting. For NKG2A-targeted delivery of an IL-2Rβγ agonist, a single molecule of de novo designed N215 endowed with Immunoglobin G(IgG)-binding ability was coupled to an antibody against NKG2A (αNKG2A) to produce αNKG2A-N215. NKG2A- and IL-2Rβγ-binding were well preserved in αNKG2A-N215, allowing αNKG2A-N215 to act as both an immune checkpoint inhibitor and a T and NK cell stimulator. Intravenously injected αNKG2A-N215 predominantly induced expansion of tumor-infiltrated CD8+ T and NK cells while showing little stimulation of Tregs. Compared with the separate combination using αNKG2A and N215, αNKG2A-N215 exerted a greater antitumor effect in mice bearing MC38 or B16/F1 tumors. 50% of mice bearing MC38 tumors were cured by αNKG2A-N215, and long-term immunological memory against the tumor was induced in these mice. These results indicate that NKG2A is another ideal target for delivery of an IL-2Rβγ agonist, and αNKG2A-N215, with specificities for both NKG2A and IL-2Rβγ, might be developed as a novel agent for immunotherapy.

IL-2 is a potent cytokine that promotes multiple immune cells proliferation and activation. Accordingly, IL-2 based immunotherapies are emerging to treat cancers or AIDS by enhancing T cell growth and function. Besides, IL-2 is indispensable in in vitro cultivation of immune cells which is a critical step of CAR-T or CAR-NK immunotherapies. In bulk manufacturing of recombinant IL-2, E. coli expression system has several advantages such as low cost and rapid growth. However, high level protein expression in prokaryotic organisms often results in inactive insoluble aggregates, also known as inclusion bodies. It is crucial and most challenging work to solubilize and refold the protein of interest from inclusion bodies to generate its bioactive form. While multiple spectrum method had been applied to monitor the refolding process, the question of how to measure the refolding efficiency from the perspective of its biological function remained unaddressed. Here we present a receptor-based sandwich ELISA method to evaluate the bioactive product in samples and provide a guidance for manufacturing bioactive rhIL-2 (recombinant human IL-2) with prokaryotic expression system. Compared to traditional antibody-based ELISA method, this novel approach truly measures the presence of functional rhIL-2 and also potentially allows the detection of rhIL-2 variants with modifications like PEGylation as it doesn't rely on the exact amino acid sequence.

Extracellular vesicles (EVs) have emerged as promising biomarkers for various diseases. Encapsulating biomolecules reflective of their parental cells, EVs are readily accessible in bodily fluids. The prospect for minimally invasive, repeatable molecular testing has stimulated significant research; however, challenges persist in isolating EVs from complex biological matrices and characterizing their limited molecular cargo. Technical advances have been pursued to address these challenges, producing innovative EV-specific platforms. This review highlights recent technological developments, focusing on EV isolation and molecular detection methodologies. Furthermore, it explores the translation of these laboratory innovations to clinical applications through the analysis of patient samples, providing insights into the potential diagnostic and prognostic utility of EV-based technologies.

The recent resurgence of Pseudorabies virus (PRV) has led to considerable economic losses in pig farms across China. As a zoonotic pathogen, the increasing number of human PRV infection cases highlights the urgent need for effective prevention and treatment strategies. Since PRV entry is initiated when glycoprotein D (gD) binds to cellular receptors such as Nectin-1, blocking this interaction presents a promising strategy for inhibiting PRV replication. While breakthroughs have been achieved with monoclonal antibodies targeting gD, mini-binders, which are small, computationally designed proteins, and can be rapidly generated by de novo design, offer an attractive alternative. In this work, 3 mini-binders, each approximately 6 kDa in size, were do novo designed targeting the hydrophobic surface of the gD protein in gD/Nectin-1 binding interface, with potent antiviral activity on PRV in PK15 cells. Notably, binder-3 exhibited the strongest binding affinity and inhibitory effect, with an IC50 value of 2.41 nM. All three mini-binders showed significant prophylactic effects, inhibiting PRV replication effectively beginning at the early stages of infection. Additionally, binder-3 show significant anti-PRV activity in a dose of 30 mg/kg at intraperitoneal administration three times daily. Our study confirmed that the mini-binders to inhibit PRV replication is a feasible scheme.

Recent advancements in immunotherapy, particularly Chimeric antigen receptor (CAR)-T cell therapy and cancer vaccines, have significantly transformed the treatment landscape for leukemia. CAR-T cell therapy, initially promising in hematologic cancers, faces notable obstacles in solid tumors due to the complex and immunosuppressive tumor microenvironment. Challenges include the heterogeneous immune profiles of tumors, variability in antigen expression, difficulties in therapeutic delivery, T cell exhaustion, and reduced cytotoxic activity at the tumor site. Additionally, the physical barriers within tumors and the immunological camouflage used by cancer cells further complicate treatment efficacy. To overcome these hurdles, ongoing research explores the synergistic potential of combining CAR-T cell therapy with cancer vaccines and other therapeutic strategies such as checkpoint inhibitors and cytokine therapy. This review describes the various immunotherapeutic approaches targeting leukemia, emphasizing the roles and interplay of cancer vaccines and CAR-T cell therapy. In addition, by discussing how these therapies individually and collectively contribute to tumor regression, this article aims to highlight innovative treatment paradigms that could enhance clinical outcomes for leukemia patients. This integrative approach promises to pave the way for more effective and durable treatment strategies in the oncology field. These combined immunotherapeutic strategies hold great promise for achieving more complete and lasting remissions in leukemia patients. Future research should prioritize optimizing treatment sequencing, personalizing therapeutic combinations based on individual patient and tumor characteristics, and developing novel strategies to enhance T cell persistence and function within the tumor microenvironment. Ultimately, these efforts will advance the development of more effective and less toxic immunotherapeutic interventions, offering new hope for patients battling this challenging disease.

Clinical trials of receptor-biased interleukin-2 (IL-2) variants in cancer therapy show limited efficacy. To investigate, we re-evaluated divergent receptor-biased IL-2 PEGylates (generated via site-specific PEGylation at residues D20 (not-β) and Y45 (not-α)), alone or in combination. Results showed the not-α variant (Y45) activates regulatory T cells (Tregs) via βγ chain binding, overriding CD8+ T cells and impairing efficacy. Conversely, the not-β IL-2 (D20) is inert alone but spatially blocks Y45's βγ engagement, suppressing Treg activation. D20 also modulates activated CD8+ T cells by preferentially binding the α chain, disrupting Y45-mediated βγ signaling to prevent exhaustion and terminal differentiation. Synergy between these PEGylates highlights the α chain as a regulatory switch reshaping Treg, CD8+ T cell, and endothelial cell fates. In syngeneic tumor models, combined therapy enhanced CD8+ T cell infiltration, suppressed tumor growth, and reduced vascular leak syndrome risk. These findings propose combinatorial IL-2 strategies targeting α chain regulation to optimize antitumor responses.

Interleukin-2 (IL-2)-based therapeutics are emerging as treatments for immunotherapy; however, systemic activation of immune cells hampers their success. Chemically controlling the activity of potent cytokines could mitigate unwanted T cell stimulation and widen their therapeutic window. In this study, we developed a strategy for the conditional activation of proteins utilizing removable peptide nucleic acid (PNA) masking groups. Site-specific installation of "Lock"-PNAs containing a cleavage thioester linkage enabled steric blockage of receptor binding sites. Rapid unmasking and activation were performed by the addition of a complementary "Key"-PNA containing a cysteine (Cys) residue, which forms a PNA-PNA duplex leading to a proximity-accelerated cleavage step and release of the active protein. We exemplified the versatility of this methodology on de novo cytokine neoleukin-2/15 (Neo-2/15) through the preparation of PNA conjugates including homodimers, PNA-stapled conjugates, and dual PNA-bridged dimers. All constructs were effectively unmasked at low micromolar concentrations. Further, we demonstrated the conditional activation of a masked conjugate of Neo-2/15 in binding studies to the IL-2 receptors and in an ex vivo T cell signaling assay displaying a 480-fold potency increase upon activation. Finally, we extended the strategy to a designed ankyrin repeat protein (DARPin) activating the human CD40 receptor demonstrating successful masking and unmasking.

In immunotherapy, peptide-based medications are showing great promise as a new class of therapies that can be used to treat autoimmune diseases, cancer, and other immune-related conditions. Peptides are being created for use in immunotherapy as vaccines, immunological modulators, and adjuvants because of their capacity to precisely alter immune responses. They can imitate endogenous signals or interact with immune cells, improving the body's capacity to identify and combat malignancies or reestablishing immunological tolerance in autoimmune disorders. Notably, peptide-based treatments have demonstrated promise in promoting tumor-specific immune responses and improving the effectiveness of already available immunotherapies, such as immune checkpoint inhibitors. Notwithstanding its potential, peptide-based medications' clinical translation is fraught with difficulties, such as those pertaining to immunogenicity, bioavailability, and peptide stability. Overcoming these obstacles has been made possible by developments in peptide engineering, including pharmacokinetic optimization, receptor-binding affinity enhancement, and the creation of innovative delivery systems. The targeted distribution and effectiveness of peptide medications can be improved by using liposomes, nanoparticles, and other delivery methods, increasing their therapeutic utility. With an emphasis on recent scientific developments, mechanisms of action, and therapeutic uses, this review examines the present status of peptide-based medications in immunotherapy. We also look at the obstacles that still need to be overcome in order to get peptide-based treatments from the lab to the clinic and offer suggestions for future research initiatives. By tackling these important problems, we hope to demonstrate how peptide-based medications have the ability to revolutionize immunotherapeutic treatment approaches.

Enzymes are essential biological macromolecules with various biological and industrial applications. As modern applications of enzymes as biocatalysts are increasingly explored, the demand for enzymes with improved catalytic properties is also increasing exponentially. Since most commercially available enzymes have a problem with long-term stability and activity under various industrial conditions, the exploration of different environments using omics technology and biotransformation of these proteins to improve stability is being recognized. Direct evolution, structure-based rational design, or de novo synthesis methods are used for enzyme engineering and developing novel enzymes with unique catalytic activity and high stability. The review provides an overview of the different classes of industrially important enzymes, their sources, and the various enzyme engineering methods used to increase their efficiency. The importance of enzyme engineering concerning the development of other techniques in the field of molecular biology is also examined.

Interleukin 2 (IL2) is a dual-acting cytokine, playing important roles in both immune activation and regulation. The role IL2 plays as a potent activator of CD8 T cells saw IL2 become one of the earliest immunotherapies, used for the treatment of cancer. In more recent years refined understanding of IL2, and the potent capacity it has for Treg stimulation, has seen low-dose IL2 therapy trialled for the treatment of auto-immune and inflammatory conditions. However, despite clinical successes, IL2 therapy is not without its caveats. The complicated receptor biology of IL2 gives rise to a narrow therapeutic window, made problematic by its short half-life. Armed with a better understanding of the structure of IL2 in complex with its receptors, many attempts have been made to create designer IL2 molecules which overcome these problems. A wide range of approaches have been used, resulting in >100 designer IL2 molecules. These include antibody complexes, fusion proteins, mutant IL2 molecules and PEGylation, each uniquely modifying the biological activity in an effort to enhance its therapeutic potential. Collectively, designer IL2 molecules form a blueprint outlining modification pathways available to other immunotherapeutics, paving the way for the next generation of immunotherapy.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. However, it continues to encounter significant obstacles, including treatment relapse and limited efficacy in solid tumors. While effector T cells exhibit robust cytotoxicity, central memory T cells and stem cell-like T cells are essential for in vivo expansion, long-term survival, and persistence. Strategies such as genetic engineering to enhance CAR-T cell efficacy and durability are often accompanied by increased safety risks, which not only raise regulatory approval thresholds but also escalate CAR-T production costs. In contrast, optimizing ex vivo manufacturing conditions represents a more straightforward and practical approach, offering the potential for rapid application to commercially approved CAR-T products and enhancement of their clinical outcomes. This review examines several factors that have been shown to improve T cell memory phenotype and in vivo cytotoxic activity, including cytokines, electrolytes, signaling pathway inhibitors, metabolic modulators, and epigenetic agents. The insights provided will guide the optimization of CAR-T cell industrial production. Furthermore, considerations for selecting appropriate conditions are discussed, balancing effectiveness, cost-efficiency, safety, and regulatory compliance while addressing current challenges in the field.

IFN-γ is a pleiotropic antiviral cytokine that coordinates innate and adaptive immune responses and induces both immunostimulatory and immunosuppressive activities, limiting its use in the clinic. Due to its antiviral role, several viruses express proteins that bind IFN-γ, blocking its interaction with the IFN-γ receptor (IFNGR). However, varicella zoster virus glycoprotein C binds IFN-γ and induces the expression of a subset of specific ISGs, similar to biased IFN-γ agonists generated based on the crystal structure of the IFN-γ - IFNGR complex. Here, we propose using structural and mechanistic information from viral proteins and biased agonists to design novel IFN-γ agonists that fine-tune IFN-γ - IFNGR activity, reducing the immunosuppressive and toxic effects of this cytokine.

Protein sequence design in the context of small molecules, nucleotides and metals is critical to enzyme and small-molecule binder and sensor design, but current state-of-the-art deep-learning-based sequence design methods are unable to model nonprotein atoms and molecules. Here we describe a deep-learning-based protein sequence design method called LigandMPNN that explicitly models all nonprotein components of biomolecular systems. LigandMPNN significantly outperforms Rosetta and ProteinMPNN on native backbone sequence recovery for residues interacting with small molecules (63.3% versus 50.4% and 50.5%), nucleotides (50.5% versus 35.2% and 34.0%) and metals (77.5% versus 36.0% and 40.6%). LigandMPNN generates not only sequences but also sidechain conformations to allow detailed evaluation of binding interactions. LigandMPNN has been used to design over 100 experimentally validated small-molecule and DNA-binding proteins with high affinity and high structural accuracy (as indicated by four X-ray crystal structures), and redesign of Rosetta small-molecule binder designs has increased binding affinity by as much as 100-fold. We anticipate that LigandMPNN will be widely useful for designing new binding proteins, sensors and enzymes.

Extracellular vesicles (EVs) are important mediators of cell-cell communication, including immune regulation. Despite the recent development of several EV-based cancer immunotherapies, their clinical efficacy remains limited. Here, we created antigen-presenting EVs to express peptide-major histocompatibility complex (pMHC) class I, costimulatory molecule and IL-2. This enabled the selective delivery of multiple immune modulators to antigen-specific CD8+ T cells, promoting their expansion in vivo without severe adverse effects. Notably, antigen-presenting EVs accumulated in the tumour microenvironment, increasing IFN-γ+ CD8+ T cell and decreasing exhausted CD8+ T cell numbers, suggesting that antigen-presenting EVs transformed the 'cold' tumour microenvironment into a 'hot' one. Combination therapy with antigen-presenting EVs and anti-PD-1 demonstrated enhanced anticancer immunity against established tumours. We successfully engineered humanized antigen-presenting EVs, which selectively stimulated tumour antigen-specific CD8+ T cells. In conclusion, engineering EVs to co-express multiple immunomodulators represents a promising method for cancer immunotherapy.

Owing to their roles in promoting T cell and natural killer (NK) cell activation and proliferation, interleukins-2 (IL-2) and interleukins-15 (IL-15) have been pursued as promising pathways to target in cancer immunotherapy. Nonetheless, their wider therapeutic application has been hampered by severe dose-limiting toxicities including systemic cytokine release and organ edema for IL-2, and inconvenient intratumoral administration for IL-15. To address these safety issues, we generated IL-2R/IL-15R×TAA (tumor-associated antigen) bispecific antibody (bsAb) pairs to selectively activate IL-2R signaling in the tumor microenvironment.

Each bsAb pair is composed of one bsAb targeting CD122 and a TAA epitope, and the other bsAb targeting CD132 and the same or a different TAA epitope. In vitro assays were performed to characterize the IL-2R/IL-15R agonistic activity of the bsAb pairs, as well as their capacity to enhance T-cell-mediated killing of TAA+ malignant cells. Using a syngeneic mouse tumor model, in vivo biological activity and systemic toxicity of the bsAb pairs were assessed in comparison with IL-2. The in vivo antitumor activity was assessed in combination with an anti-mouse programmed cell death protein 1 (mPD-1) monoclonal antibody.

We demonstrated with two different TAAs (human epidermal growth factor receptor 2 (HER2) and mesothelin (MSLN)) that the CD122×TAA/CD132×TAA bsAb pairs mediate effective activation of immune cells exclusively in the presence of TAA+ tumor cells. In syngeneic hMSLN-MC38 tumor-bearing mice, the CD122×MSLN-1/CD132×MSLN-2 bsAb pair promotes selective activation and expansion of NK cells and central memory CD8+ T cells inside the tumor without inducing organ edema or systemic cytokine release, two well-known manifestations of IL-2 associated toxicity. In combination with checkpoint inhibitor anti-mPD-1, the bsAb pair boosts the accumulation of CD8+ effector T cells and NK cells, leading to a favorable CD8+ T cell to CD4+ regulatory T cell ratio for a more robust inhibition of tumor growth.

Overall, the findings suggest that this innovative therapeutic approach effectively leverages the antitumor activity of IL-2 and IL-15 pathways while minimizing their associated systemic toxicities. This dual bsAb format holds potential for broader application in other immune-activating pathways.

The pleotropic nature of interleukin-2 (IL2) has allowed it to be used as both a pro-inflammatory and anti-inflammatory therapeutic agent, through promotion of regulatory T cell (Treg) responses via the trimeric IL2RABG receptor or promotion of CD8 T cell responses via the dimeric IL2RBG receptor, respectively. However, the utility of IL2 as a treatment is limited by this same pleiotropy, and protein engineering to bias specificity towards either Treg or CD8 T cell lineage often requires a trade-off in protein production or total bioactivity. Here we use SolubiS and dTANGO, computational algorithm-based methods, to predict mutations within the IL2 structure to improve protein production yield in muteins with altered cellular selectivity, to generate combined muteins with elevated therapeutic potential. The design and testing process identified the V106R (murine) / V91R (human) mutation as a Treg-enhancing mutein, creating a cation repulsion to inhibit primary binding to IL2RB, with a post-IL2RA confirmational shift enabling secondary IL2RB binding, and hence allowing the trimeric receptor complex to form. In human IL2, additional N90R T131R aggregation-protecting mutations could improve protein yield of the V91R mutation. The approach also generated novel CD8 T cell-promoting mutations. Y59K created a cation-cation repulsion with IL2RA, while Q30W enhanced CD8 T cell activity through potential π-stacking enhancing binding to IL2RB, with the combination highly stimulatory for CD8 T cells. For human IL2, Y45K (homolog to murine Y59K) coupled with E62K prevented IL2RA binding, however it required the aggregation-protecting mutations of N90R T131R to rescue production. These muteins, designed with both cellular specificity and protein production features, have potential as both biological tools and therapeutics.

Adoptive transfer of chimeric antigen receptor (CAR)-modified natural killer (NK) cells represents a transformative approach that has significantly advanced clinical outcomes in patients with malignant hematological conditions. However, the efficacy of CAR-NK cells in treating solid tumors is limited by their exhaustion, impaired infiltration and poor persistence in the immunosuppressive tumor microenvironment (TME). As NK cell functional states are associated with IL-2 cascade, we engineered mesothelin-specific CAR-NK cells that secrete neoleukin-2/15 (Neo-2/15), an IL-2Rβγ agonist, to resist immunosuppressive polarization within TME. The adoptively transferred Neo-2/15-armored CAR-NK cells exhibited enhanced cytotoxicity, less exhaustion and longer persistence within TME, thereby having superior antitumor activity against pancreatic cancer and ovarian cancer. Mechanistically, Neo-2/15 provided sustained and enhanced downstream IL-2 receptor signaling, which promotes the expression of c-Myc and nuclear respiratory factor 1 (NRF1) in CAR-NK cells. This upregulation was crucial for maintaining mitochondrial adaptability and metabolic resilience, ultimately leading to increased cytotoxicity and pronounced persistence of CAR-NK cells within the TME. The resistance against TME immunosuppressive polarization necessitated the upregulation of NRF1, which is essential to the augmentative effects elicited by Neo-2/15. Overexpression of NRF1 significantly bolsters the antitumor efficacy of CAR-NK cells both in vitro and in vivo, with increased ATP production. Collectively, Neo-2/15-expressing CAR-NK cells exerts superior antitumor effects by exhaustion-resistance and longer survival in solid tumors.

Immune receptors have emerged as critical therapeutic targets for cancer immunotherapy. Designed protein binders can have high affinity, modularity, and stability and hence could be attractive components of protein therapeutics directed against these receptors, but traditional Rosetta based protein binder methods using small globular scaffolds have difficulty achieving high affinity on convex targets. Here we describe the development of helical concave scaffolds tailored to the convex target sites typically involved in immune receptor interactions. We employed these scaffolds to design proteins that bind to TGFβRII, CTLA-4, and PD-L1, achieving low nanomolar to picomolar affinities and potent biological activity following experimental optimization. Co-crystal structures of the TGFβRII and CTLA-4 binders in complex with their respective receptors closely match the design models. These designs should have considerable utility for downstream therapeutic applications.

Human interleukin-2 (IL-2) stimulates the differentiation and expansion of diverse immune cells dose-dependently. As an immunotherapy agent to treat metastatic cancers, IL-2 has been used in clinical practice and has demonstrated clear antitumor effects; however, its short half-life, the risk of capillary leak syndrome, and the unintended activation of immunosuppressive Treg cells hinder its clinical application. To address these challenges, a novel PEGylated interleukin-2 analogue, SHR-1916, was designed. Its cellular selectivity, efficacy, and improved pharmacokinetic profiles were investigated.

The binding affinities were characterized by surface plasmon resonance (SPR) in vitro. Subsequently, the stimulatory properties were investigated in a murine cell line (CTLL-2), a human cell line (M07e), and human peripheral blood mononuclear cells (PBMCs). To assess the anti-tumor efficacy, a CT-26 colon carcinoma syngeneic model in BALB/c mice and a A375 human melanoma xenograft model using PBMC humanized NCG mice were used in vivo. Moreover, the pharmacokinetic behavior following a single intravenous or subcutaneous dose was evaluated in Sprague-Dawley rats.

SHR-1916 abolished binding to its receptor IL-2Rα, as evidenced by SPR assays, and exerted its activity mainly through binding to IL-2Rβγ, as confirmed by CTLL-2 and M07e cell proliferation assays. In contrast to IL-2, SHR-1916 exhibited a more biased activation of CD8+ T and NK cells compared to Treg cells and stimulated an increase in IFNγ secretion in PBMCs dose-dependently without triggering the release of other potential side effect-associated cytokines. In CT26 colon carcinoma and A375 melanoma models, SHR-1916 significantly reduced the tumor burden. Pharmacokinetic results showed that SHR-1916 had a significantly prolonged half-life in rats.

SHR-1916 exhibited excellent cellular selectivity, anti-tumor efficacies, and improved pharmacokinetics. It has the potential to serve as a novel immunotherapeutic agent designed to enhance IL-2's immune-stimulating activities and promote its tolerability while reducing the immunoregulatory function of Treg cells.

Affinity proteins based on a three-helix bundle (affibodies, alphabodies, and computationally de novo designed ones) have been shown to be a general platform to discover binders with properties reminiscent of antibodies, combining high target specificity with affinities reaching well below the nanomolar. Herein, we report a strategy, coined self-assembled proteomimetic (SAP), to mimic such three-helix bundle architecture with a hybridization-enforced two-helix coiled coil that is obtained by templated native chemical ligation (T-NCL) of PNA-peptide conjugates. This SAP strategy stands out by its synthetic accessibility, reducing the length on the longest synthetic peptide to less than 30 amino acids which is readily attainable by standard SPPS methodologies. We show that the T-NCL dramatically accelerates the ligation, enabling this chemistry to proceed in a combinatorial fashion at low micromolar concentrations. We demonstrate that small combinatorial libraries of SAPs can be prepared in one operation and used directly in affinity selections against a target of interest with an LC-MS analysis of the fittest binders. Moreover, we show that the underlying design paradigm is functional for SAPs based on structurally distinct three-helix peptides aimed at different therapeutic targets, namely HER2 and spike's RBD, reaching picomolar affinities. We further illustrate that the affinity of the SAP can be allosterically regulated using a toehold displacement of the hybridizing PNAs to disrupt the coiled coil stabilization. Finally, we show that an RBD-targeting SAP effectively inhibits viral entry of SARS-CoV-2 with an IC50 of 2.8 nM.

Peptides can bind to specific sites on larger proteins and thereby function as inhibitors and regulatory elements. Peptide fragments of larger proteins are particularly attractive for achieving these functions due to their inherent potential to form native-like binding interactions. Recently developed experimental approaches allow for high-throughput measurement of protein fragment inhibitory activity in living cells. However, it has thus far not been possible to predict de novo which of the many possible protein fragments bind to protein targets, let alone act as inhibitors. We have developed a computational method, FragFold, that employs AlphaFold to predict protein fragment binding to full-length proteins in a high-throughput manner. Applying FragFold to thousands of fragments tiling across diverse proteins revealed peaks of predicted binding along each protein sequence. Comparisons with experimental measurements establish that our approach is a sensitive predictor of fragment function: Evaluating inhibitory fragments from known protein-protein interaction interfaces, we find 87% are predicted by FragFold to bind in a native-like mode. Across full protein sequences, 68% of FragFold-predicted binding peaks match experimentally measured inhibitory peaks. Deep mutational scanning experiments support the predicted binding modes and uncover superior inhibitory peptides in high throughput. Further, FragFold is able to predict previously unknown protein binding modes, explaining prior genetic and biochemical data. The success rate of FragFold demonstrates that this computational approach should be broadly applicable for discovering inhibitory protein fragments across proteomes.

Identification of effective therapies for colorectal cancer (CRC) remains an urgent medical need, especially for the microsatellite stable (MSS) phenotype. In our previous study, potassium oxonate (PO), a uricase inhibitor commonly used for elevating uric acid in mice, unexpectedly showed remarkable inhibition of tumor growth when combined with anti-programmed death-1 (PD-1). Further research demonstrated that the combination of potassium oxonate and anti-PD-1 could reprogram the immune microenvironment. This study aimed to explore the anti-tumor effect of PO combined with anti-PD-1, and investigate the impact on the immunosuppressive tumor microenvironment (TME).

We established a syngeneic mouse model of CRC and divided into groups of control group, single drugs group of PO and anti-PD-1, and the combination group. Use the HE staining, immunohistochemistry (IHC) and TUNEL staining of tumor issues to verify the anti-neoplasm of each group. We also tested the changes of TME through flow cytometry of spleen of mice in each group, as well as the IHC of cytokines.

The co-therapy of PO and anti-PD-1 showed admirable anti-tumor effect compared with the control group and the single drug groups. The TME were tended to an environment beneficial for killing tumors by enhancing chemotactic factor release, increasing CD8+ T cell infiltration and activation, and decreasing the amount of regulatory T cells. Moreover, IFN-γ and IL-2 secretion were found to be enriched in the tumor TME.

Our study indicated that combination of PO and anti-PD-1 could synergistically suppress CRC progression and altered the tumor microenvironment in favor of antitumor immune responses.

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.

Toll-like Receptor 3 (TLR3) is a pattern recognition receptor that initiates antiviral immune responses upon binding double-stranded RNA (dsRNA). Several nucleic acid-based TLR3 agonists have been explored clinically as vaccine adjuvants in cancer and infectious disease, but present substantial manufacturing and formulation challenges. Here, we use computational protein design to create novel miniproteins that bind to human TLR3 with nanomolar affinities. Cryo-EM structures of two minibinders in complex with TLR3 reveal that they bind the target as designed, although one partially unfolds due to steric competition with a nearby N-linked glycan. Multivalent forms of both minibinders induce NF-κB signaling in TLR3-expressing cell lines, demonstrating that they may have therapeutically relevant biological activity. Our work provides a foundation for the development of specific, stable, and easy-to-formulate protein-based agonists of TLRs and other pattern recognition receptors.

Bacterial outer membrane vesicles (OMVs) have emerged as promising vehicles for anticancer drug delivery due to their inherent tumor tropism, immune-stimulatory properties, and potential for functionalization with therapeutic proteins. Despite their advantages, the high lipopolysaccharide (LPS) endotoxin content in the OMVs raises significant safety and regulatory challenges. In this work, we produce LPS-attenuated and LPS-free OMVs and systematically assess the effects of LPS modification on OMVs' physicochemical characteristics, membrane protein content, immune-stimulatory capacity, tolerability, and anticancer efficacy. Our findings reveal that LPS removal increased the maximal tolerated dose of the OMVs by over 25-fold. When adjusted for comparable safety profiles, LPS-free OMVs exhibit superior anticancer effects compared with wild-type OMVs. Mechanistic investigations indicate that the LPS removal obviates immune cell death caused by LPS and reduces the negatory effects of wild type of OMVs on tumor immune cell infiltrates. We further show the functionality of the LPS-free OMV through the incorporation of an IL-2 variant protein (Neo-2/15). This functionalization augments OMV's ability of the OMV to inhibit tumor growth and promote lymphocyte infiltration into the tumor microenvironment. This study presents a safe and functionalizable OMV with improved translational prospect.

Human interleukin-2 (IL-2) is a crucial cytokine for T cell regulation, with therapeutic potential in cancer and autoimmune diseases. However, IL-2's pleiotropic effects across different immune cell types often lead to toxicity and limited efficacy. Previous efforts to enhance IL-2's therapeutic profile have focused on modifying its receptor binding sites. Yet, the underlying dynamics and intramolecular networks contributing to IL-2 receptor recognition remain unexplored. This study presents a detailed characterization of IL-2 dynamics compared to two engineered IL-2 mutants, "superkines" S15 and S1, which exhibit biased signaling towards effector T cells. Using NMR spectroscopy and molecular dynamics simulations, we demonstrate significant variations in core dynamic pathways and conformational exchange rates across these three IL-2 variants. We identify distinct allosteric networks and minor state conformations in the superkines, despite their structural similarity to wild-type IL-2. Furthermore, we rationally design a mutation (L56A) in the S1 superkine's core network, which partially reverts its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. Our results reveal that IL-2 superkine core dynamics play a critical role in their enhanced receptor binding and function, suggesting that modulating IL-2 dynamics and core allostery represents an untapped approach for designing immunotherapies with improved immune cell selectivity profiles.

Cytokines are proteins used by immune cells to communicate with each other and with cells in their environment. The pleiotropic effects of cytokine networks are determined by which cells express cytokines and which cells express cytokine receptors, with downstream outcomes that can differ based on cell type and environmental cues. Certain cytokines, such as interferon (IFN)-γ, have been clearly linked to anti-tumor immunity, while others, such as the innate inflammatory cytokines, promote oncogenesis. Here we provide an overview of the functional roles of cytokines in the tumor microenvironment. Although we have a sophisticated understanding of cytokine networks, therapeutically targeting cytokine pathways in cancer has been challenging. We discuss current progress in cytokine blockade, cytokine-based therapies, and engineered cytokine therapeutics as emerging cancer treatments of interest.

Synthetic binding proteins (SBPs) represent a pivotal class of artificially engineered proteins, meticulously crafted to exhibit targeted binding properties and specific functions. Here, the SYNBIP database, a comprehensive resource for SBPs, has been significantly updated. These enhancements include (i) featuring 3D structures of 899 SBP-target complexes to illustrate the binding epitopes of SBPs, (ii) using the structures of SBPs in the monomer or complex forms with target proteins, their sequence space has been expanded five times to 12 025 by integrating a structure-based protein generation framework and a protein property prediction tool, (iii) offering detailed information on 78 473 newly identified SBP-like scaffolds from the RCSB Protein Data Bank, and an additional 16 401 555 ones from the AlphaFold Protein Structure Database, and (iv) the database is regularly updated, incorporating 153 new SBPs. Furthermore, the structural models of all SBPs have been enhanced through the application of the AlphaFold2, with their clinical statuses concurrently refreshed. Additionally, the design methods employed for each SBP are now prominently featured in the database. In sum, SYNBIP 2.0 is designed to provide researchers with essential SBP data, facilitating their innovation in research, diagnosis and therapy. SYNBIP 2.0 is now freely accessible at https://idrblab.org/synbip/.

Proteins have proven to be useful agents in a variety of fields, from serving as potent therapeutics to enabling complex catalysis for chemical manufacture. However, they remain difficult to design and are instead typically selected for using extensive screens or directed evolution. Recent developments in protein large language models have enabled fast generation of diverse protein sequences in unexplored regions of protein space predicted to fold into varied structures, bind relevant targets, and catalyze novel reactions. Nevertheless, we lack methods to characterize these proteins experimentally at scale and update generative models based on those results. We describe Protein CREATE (Computational Redesign via an Experiment-Augmented Training Engine), an integrated computational and experimental pipeline that incorporates an experimental workflow leveraging next generation sequencing and phage display with single-molecule readouts to collect vast amounts of quantitative binding data for updating protein large language models. We use Protein CREATE to generate and assay thousands of designed binders to IL-7 receptor α and insulin receptor with parallel positive and negative selections to identify on-target binders. We discover not only individual novel binders but also features of ligand-receptor binding, including preservation of the IL7Rα - ligand hydrophobic interface specifically and existence of multiple approaches to contact the insulin receptor. We also demonstrate the importance of structural features, such as the lack of unpaired cysteine residues, toward design fidelity and find computational pre-screening metrics, such as interchain predicted TM scoring (iPTM), while useful, are imperfect predictors as they neither guarantee experimental binding nor rule it out. We use the data collected from Protein CREATE to score designs from the initial generative models. Globally, Protein CREATE will power future closed-loop design-build-test cycles to enable fine-grained design of protein binders.

Voltage-gated ion channels (VGICs) are pivotal in regulating electrical activity in excitable cells and are critical pharmaceutical targets for treating many diseases including cardiac arrhythmia and neuropathic pain. Despite their significance, challenges such as achieving target selectivity persist in VGIC drug development. Recent progress in deep learning, particularly diffusion models, has enabled the computational design of protein binders for any clinically relevant protein based solely on its structure. These developments coincide with a surge in experimental structural data for VGICs, providing a rich foundation for computational design efforts. This review explores the recent advancements in computational protein design using deep learning and diffusion methods, focusing on their application in designing protein binders to modulate VGIC activity. We discuss the potential use of these methods to computationally design protein binders targeting different regions of VGICs, including the pore domain, voltage-sensing domains, and interface with auxiliary subunits. We provide a comprehensive overview of the different design scenarios, discuss key structural considerations, and address the practical challenges in developing VGIC-targeting protein binders. By exploring these innovative computational methods, we aim to provide a framework for developing novel strategies that could significantly advance VGIC pharmacology and lead to the discovery of effective and safe therapeutics.