Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models.

BACKGROUND: Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS: Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS: Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS: Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Selenium chemistry for spatio-selective peptide and protein functionalization.

The ability to construct a peptide or protein in a spatio-specific manner is of great interest for therapeutic and biochemical research. However, the various functional groups present in peptide sequences and the need to perform chemistry under mild and aqueous conditions make selective protein functionalization one of the greatest synthetic challenges. The fascinating paradox of selenium (Se) - being found in both toxic compounds and also harnessed by nature for essential biochemical processes - has inspired the recent exploration of selenium chemistry for site-selective functionalization of peptides and proteins. In this Review, we discuss such approaches, including metal-free and metal-catalysed transformations, as well as traceless chemical modifications. We report their advantages, limitations and applications, as well as future research avenues.

Overexpression of Synoviolin and miR-125a-5p, miR-19b-3p in peripheral blood of rheumatoid arthritis patients after treatment with conventional DMARDs and methylprednisolone.

PURPOSE: SYVN1 is an endoplasmic reticulum (ER)-resident E3 ubiquitin ligase that has an essential function along with SEL1L in rheumatoid arthritis (RA) pathogenesis. This study aimed to investigate the changes in the expression of peripheral blood ncRNAs and SYVN1-SEL1L affected by DMARDs treatment. METHODS: Twenty-five newly diagnosed RA patients were randomly assigned to receive conventional DMARDs (csDMARDs) and methylprednisolone for six months. The peripheral blood gene expression of SYVN1 and SEL1L and possible regulatory axes, NEAT1, miR-125a-5p, and miR-19b-3p, were evaluated before and after qRT-PCR. We also compared differences between the patients and healthy controls (HCs), and statistical analyses were performed to determine the correlation between ncRNAs with SYVN1-SEL1L and the clinical parameters of RA. RESULTS: Expression of NEAT1 (P = 0.0001), miR-19b-3p (P = 0.007), miR-125a-5p (P = 0.005), and SYVN1 (P = 0.036) was significantly increased in newly diagnosed patients compared to HCs; also, miR-125a-5p, miR-19b-3p, and SYVN1 were significantly overexpressed after treatment (P = 0.001, P = 0.001, and P = 0.005, respectively). NEAT1 was positively correlated with SYVN1, and miR-125a-5p had a negative correlation with anti-cyclic citrullinated peptides. The ROC curve analysis showed the potential role of selected ncRNAs in RA pathogenesis. CONCLUSION: The results indicate the ineffectiveness of the csDMARDs in reducing SYVN1 expression. The difference in expression of ncRNAs might be useful markers for monitoring disease activity and determining therapeutic responses in RA patients. Key Points • The expression of NEAT1 is significantly upregulated in RA patients compared to HC subjects. • miR-19b-3p, miR-125a-5p, and SYVN1 are significantly upregulated in RA patients compared to HC subjects. • The expression of miR-19b-3p and miR-125a-5p is significantly increased in RA patients after treatment with DMARDs and methylprednisolone. • NEAT1 is positively correlated with SYVN1.

Bioconjugation Techniques for Enhancing Stability and Targeting Efficiency of Protein and Peptide Therapeutics.

Bioconjugation techniques have emerged as powerful tools for enhancing the stability and targeting efficiency of protein and peptide therapeutics. This review provides a comprehensive analysis of the various bioconjugation strategies employed in the field. The introduction highlights the significance of bioconjugation techniques in addressing stability and targeting challenges associated with protein and peptide-based drugs. Chemical and enzymatic bioconjugation methods are discussed, along with crosslinking strategies for covalent attachment and site-specific conjugation approaches. The role of bioconjugation in improving stability profiles is explored, showcasing case studies that demonstrate successful stability enhancement. Furthermore, bioconjugation techniques for ligand attachment and targeting are presented, accompanied by examples of targeted protein and peptide therapeutics. The review also covers bioconjugation approaches for prolonging circulation and controlled release, focusing on strategies to extend half-life, reduce clearance, and design-controlled release systems. Analytical characterization techniques for bioconjugates, including the evaluation of conjugation efficiency, stability, and assessment of biological activity and targeting efficiency, are thoroughly examined. In vivo considerations and clinical applications of bioconjugated protein and peptide therapeutics, including pharmacokinetic and pharmacodynamic considerations, as well as preclinical and clinical developments, are discussed. Finally, the review concludes with an overview of future perspectives, emphasizing the potential for novel conjugation methods and advanced targeting strategies to further enhance the stability and targeting efficiency of protein and peptide therapeutics.

Polypeptides as alternatives to PEGylation of therapeutic agents.

INTRODUCTION: Due to the concerns raised by the extensive application of PEGylation, polypeptides have stood out as excellent candidates with adequate biocompatibility and biodegradability with tunable hydrophilicity. AREAS COVERED: In this review, polypeptides with the potential to replace PEGylation have been summarized and their application has been reviewed, including XTEN, PASylation, polysarcosine, zwitterion polypeptides, ELPylation, etc. Besides their strengths, the remaining challenges have also been discussed and the future perspectives have been provided. EXPERT OPINION: Polypeptides have been applied in the designing of peptide/protein drugs as well as nanomedicines, and some of the pharmaceutics have made it into the clinical trials and got approved. These polypeptides showed similar hydrophilic properties to PEGylation, which increased the hydrodynamic volumes of protein drugs, reduced kidney elimination, decreased protein-polymer interaction and potentially improved the drug delivery efficiency due to the extended circulation time in the system. Moreover, they demonstrated superior biodegradability and biocompatibility, compensating for the deficiencies for polymers such as PEG.

Potential Application of Self-Assembled Peptides and Proteins in Breast Cancer and Cervical Cancer.

Ongoing research is gradually broadening the idea of cancer treatment, with attention being focused on nanoparticles to improve the stability, therapeutic efficacy, targeting, and other important metrics of conventional drugs and traditional drug delivery methods. Studies have demonstrated that drug delivery carriers based on biomaterials (e.g., protein nanoparticles and lipids) and inorganic materials (e.g., metal nanoparticles) have potential anticancer effects. Among these carriers, self-assembled proteins and peptides, which are highly biocompatible and easy to standardize and produce, are strong candidates for the preparation of anticancer drugs. Breast cancer (BC) and cervical cancer (CC) are two of the most common and deadly cancers in women. These cancers not only threaten lives globally but also put a heavy burden on the healthcare system. Despite advances in medical care, the incidence of these two cancers, particularly CC, which is almost entirely preventable, continues to rise, and the mortality rate remains steady. Therefore, there is still a need for in-depth research on these two cancers to develop more targeted, efficacious, and safe therapies. This paper reviews the types of self-assembling proteins and peptides (e.g., ferritin, albumin, and virus-like particles) and natural products (e.g., soy and paclitaxel) commonly used in the treatment of BC and CC and describes the types of drugs that can be delivered using self-assembling proteins and peptides as carriers (e.g., siRNAs, DNA, plasmids, and mRNAs). The mechanisms (including self-assembly) by which the natural products act on CC and BC are discussed. The mechanism of action of natural products on CC and BC and the mechanism of action of self-assembled proteins and peptides have many similarities (e.g., NF-KB and Wnt). Thus, natural products using self-assembled proteins and peptides as carriers show potential for the treatment of BC and CC.

Therapeutic Fusion Proteins.

Therapeutic fusion proteins are a class of hybrid constructs that combine distinct biomolecules into a single platform with the additive effects of the components. The ability to fuse two unrelated proteins provides a means to localize mechanisms to better treat a range of diseases. Fusion proteins can be designed to impart diverse functions, including increasing half-life, providing targeting, and enabling sustained signaling. Of these, half-life extenders, which are fused to a therapeutic protein to increase exposure, are the most established group of fusion proteins, with many clinical successes. Rapid advances in antibody and antibody-derivative technology have enabled the fusion of targeting domains with therapeutic proteins. An emerging group of therapeutic fusion proteins has two separate active functions. Although most research for therapeutic fusion proteins focuses on cancer, prior successes provide a foundation for studies into other diseases as well. The exponential emergence of biopharmaceuticals gives precedence for increased research into therapeutic fusion proteins for a multitude of diseases.

The BET degrader ZBC260 suppresses stemness and tumorigenesis and promotes differentiation in triple-negative breast cancer by disrupting inflammatory signaling.

BACKGROUND: Breast cancer stem cells (BCSCs) are resistant to standard therapies, facilitate tumor dissemination, and contribute to relapse and progression. Super-enhancers are regulators of stemness, and BET proteins, which are critical for super-enhancer function, are a potential therapeutic target. Here, we investigated the effects of BET proteins on the regulation of breast cancer stemness using the pan-BET degrader ZBC260. METHODS: We evaluated the effect of ZBC260 on CSCs in TNBC cell lines. We assessed the effect of ZBC260 on cellular viability and tumor growth and measured its effects on cancer stemness. We used RNA sequencing and stemness index to determine the global transcriptomic changes in CSCs and bulk cells and further validated our findings by qPCR, western blot, and ELISA. RESULTS: ZBC260 potently inhibited TNBC growth both in vitro and in vivo. ZBC260 reduced stemness as measured by cell surface marker expression, ALDH activity, tumorsphere number, and stemness index while increasing differentiated cells. GSEA analysis indicated preferential downregulation of stemness-associated and inflammatory genes by ZBC260 in ALDH+ CSCs. CONCLUSIONS: The BET degrader ZBC260 is an efficient degrader of BET proteins that suppresses tumor progression and decreases CSCs through the downregulation of inflammatory genes and pathways. Our findings support the further development of BET degraders alone and in combination with other therapeutics as CSC targeting agents.

Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis.

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.

Polysaccharides, proteins, and synthetic polymers based multimodal hydrogels for various biomedical applications: A review.

Nature-derived or biologically encouraged hydrogels have attracted considerable interest in numerous biomedical applications owing to their multidimensional utility and effectiveness. The internal architecture of a hydrogel network, the chemistry of the raw materials involved, interaction across the interface of counter ions, and the ability to mimic the extracellular matrix (ECM) govern the clinical efficacy of the designed hydrogels. This review focuses on the mechanistic viewpoint of different biologically driven/inspired biomacromolecules that encourages the architectural development of hydrogel networks. In addition, the advantage of hydrogels by mimicking the ECM and the significance of the raw material selection as an indicator of bioinertness is deeply elaborated in the review. Furthermore, the article reviews and describes the application of polysaccharides, proteins, and synthetic polymer-based multimodal hydrogels inspired by or derived from nature in different biomedical areas. The review discusses the challenges and opportunities in biomaterials along with future prospects in terms of their applications in biodevices or functional components for human health issues. This review provides information on the strategy and inspiration from nature that can be used to develop a link between multimodal hydrogels as the main frame and its utility in biomedical applications as the primary target.

Self-assembled block copolymer biomaterials for oral delivery of protein therapeutics.

Protein therapeutics have guided a transformation in disease treatment for various clinical conditions. They have been successful in numerous applications, but administration of protein therapeutics has been limited to parenteral routes which can decrease patient compliance as they are invasive and painful. In recent years, the synergistic relationship of novel biomaterials with modern protein therapeutics has been crucial in the treatment of diseases that were once thought of as incurable. This has guided the development of a variety of alternative administration routes, but the oral delivery of therapeutics remains one of the most desirable due to its ease of administration. This review addresses important aspects of micellar structures prepared by self-assembled processes with applications for oral delivery. These two characteristics have not been placed together in previous literature within the field. Therefore, we describe the barriers for delivery of protein therapeutics, and we concentrate in the oral/transmucosal pathway where drug carriers must overcome several chemical, physical, and biological barriers to achieve a successful therapeutic effect. We critically discuss recent research on biomaterials systems for delivering such therapeutics with an emphasis on self-assembled synthetic block copolymers. Polymerization methods and nanoparticle preparation techniques are similarly analyzed as well as relevant work in this area. Based on our own and others' research, we analyze the use of block copolymers as therapeutic carriers and their promise in treating a variety of diseases, with emphasis on self-assembled micelles for the next generation of oral protein therapeutic systems.

Noncovalent PEGylation of protein and peptide therapeutics.

Clinical applications of protein therapeutics-an advanced generation of drugs characterized by high biological specificity-are rapidly expanding. However, their development is often impeded by unfavorable pharmacokinetic profiles and largely relies on the use of drug delivery systems to prolong their in vivo half-life and suppress undesirable immunogenicity. Although a commercially established PEGylation technology based on protein conjugation with poly(ethylene glycol) (PEG)-protective steric shield resolves some of the challenges, the search for alternatives continues. Noncovalent PEGylation, which mainly relies on multivalent (cooperative) interactions and high affinity (host-guest) complexes formed between protein and PEG offers a number of potential advantages. Among them are dynamic or reversible protection of the protein with minimal loss of biological activity, drastically lower manufacturing costs, "mix-and-match" formulations approaches, and expanded scope of PEGylation targets. While a great number of innovative chemical approaches have been proposed in recent years, the ability to effectively control the stability of noncovalently assembled protein-PEG complexes under physiological conditions presents a serious challenge for the commercial development of the technology. In an attempt to identify critical factors affecting pharmacological behavior of noncovalently linked complexes, this Review follows a hierarchical analysis of various experimental techniques and resulting supramolecular architectures. The importance of in vivo administration routes, degradation patterns of PEGylating agents, and a multitude of potential exchange reactions with constituents of physiological compartments are highlighted. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Intracellular protein delivery: New insights into the therapeutic applications and emerging technologies.

The inability to cross the plasma membranes traditionally limited the therapeutic use of recombinant proteins. However, in the last two decades, novel technologies made delivering proteins inside the cells possible. This allowed researchers to unlock intracellular targets, once considered 'undruggable', bringing a new research area to emerge. Protein transfection systems display a large potential in a plethora of applications. However, their modality of action is often unclear, and cytotoxic effects are elevated, whereas experimental conditions to increase transfection efficacy and cell viability still need to be identified. Furthermore, technical complexity often limits in vivo experimentation, while challenging industrial and clinical translation. This review highlights the applications of protein transfection technologies, and then critically discuss the current methodologies and their limitations. Physical membrane perforation systems are compared to systems exploiting cellular endocytosis. Research evidence of the existence of either extracellular vesicles (EVs) or cell-penetrating peptides (CPPs)- based systems, that circumvent the endosomal systems is critically analysed. Commercial systems, novel solid-phase reverse protein transfection systems, and engineered living intracellular bacteria-based mechanisms are finally described. This review ultimately aims at finding new methodologies and possible applications of protein transfection systems, while helping the development of an evidence-based research approach.

Bromodomain and Extraterminal Domain (BET) Protein Inhibition Hinders Glioblastoma Progression by Inducing Autophagy-Dependent Differentiation.

Glioblastoma multiforme (GBM) is the most common and aggressive type of malignant primary brain tumor, and it is characterized by a high recurrence incidence and poor prognosis due to the presence of a highly heterogeneous mass of stem cells with self-renewal capacity and stemness maintenance ability. In recent years, the epigenetic landscape of GBM has been explored and many epigenetic alterations have been investigated. Among the investigated epigenetic abnormalities, the bromodomain and extra-terminal domain (BET) chromatin readers have been found to be significantly overexpressed in GBM. In this work, we investigated the effects of BET protein inhibition on GBM cell reprogramming. We found that the pan-BET pharmacological inhibitor JQ1 was able to promote a differentiation program in GBM cells, thus impairing cell proliferation and enhancing the toxicity of the drug Temozolomide (TMZ). Notably, the pro-differentiation capability of JQ1 was prevented in autophagy-defective models, suggesting that autophagy activation is necessary for BET protein activity in regulating glioma cell fate. Given the growing interest in epigenetic therapy, our results further support the possibility of introducing a BET-based approach in GBM clinical management.

Engineering protein-based therapeutics through structural and chemical design.

Protein-based therapeutics have led to new paradigms in disease treatment. Projected to be half of the top ten selling drugs in 2023, proteins have emerged as rivaling and, in some cases, superior alternatives to historically used small molecule-based medicines. This review chronicles both well-established and emerging design strategies that have enabled this paradigm shift by transforming protein-based structures that are often prone to denaturation, degradation, and aggregation in vitro and in vivo into highly effective therapeutics. In particular, we discuss strategies for creating structures with increased affinity and targetability, enhanced in vivo stability and pharmacokinetics, improved cell permeability, and reduced amounts of undesired immunogenicity.

Smart Nanosystems for Overcoming Multiple Biological Barriers in Cancer Nanomedicines Transport: Design Principles, Progress, and Challenges.

The development of smart nanosystems, which could overcome diverse biological barriers of nanomedicine transport, has received intense scientific interest in improving the therapeutic efficacies of traditional nanomedicines. However, the reported nanosystems generally hold disparate structures and functions, and the knowledge of involved biological barriers is usually scattered. There is an imperative need for a summary of biological barriers and how these smart nanosystems conquer biological barriers, to guide the rational design of the new-generation nanomedicines. This review starts from the discussion of major biological barriers existing in nanomedicine transport, including blood circulation, tumoral accumulation and penetration, cellular uptake, drug release, and response. Design principles and recent progress of smart nanosystems in overcoming the biological barriers are overviewed. The designated physicochemical properties of nanosystems can dictate their functions in biological environments, such as protein absorption inhibition, tumor accumulation, penetration, cellular internalization, endosomal escape, and controlled release, as well as modulation of tumor cells and their resident tumor microenvironment. The challenges facing smart nanosystems on the road heading to clinical approval are discussed, followed by the proposals that could further advance the nanomedicine field. It is expected that this review will provide guidelines for the rational design of the new-generation nanomedicines for clinical use.

Delivering on the promise of protein degraders.

Over the past 3 years, the first bivalent protein degraders intentionally designed for targeted protein degradation (TPD) have advanced to clinical trials, with an initial focus on established targets. Most of these clinical candidates are designed for oral administration, and many discovery efforts appear to be similarly focused. As we look towards the future, we propose that an oral-centric discovery paradigm will overly constrain the chemical designs that are considered and limit the potential to drug novel targets. In this Perspective, we summarize the current state of the bivalent degrader modality and propose three categories of degrader designs, based on their likely route of administration and requirement for drug delivery technologies. We then describe a vision for how parenteral drug delivery, implemented early in research and supported by pharmacokinetic-pharmacodynamic modelling, can enable exploration of a broader drug design space, expand the scope of accessible targets and deliver on the promise of protein degraders as a therapeutic modality.

Supramolecular Phenylalanine-Derived Hydrogels for the Sustained Release of Functional Proteins.

Protein-based therapeutics have emerged as next-generation pharmaceutical agents for oncology, bone regeneration, autoimmune disorders, viral infections, and other diseases. The clinical application of protein therapeutics has been impeded by pharmacokinetic and pharmacodynamic challenges including off-target toxicity, rapid clearance, and drug stability. Strategies for the localized and sustained delivery of protein therapeutics have shown promise in addressing these challenges. Hydrogels are critical materials that enable these delivery strategies. Supramolecular hydrogels composed of self-assembled materials have demonstrated biocompatibility advantages over polymer hydrogels, with peptide and protein-based gels showing strong potential. However, cost is a significant drawback of peptide-based supramolecular hydrogels. Supramolecular hydrogels composed of inexpensive low-molecular-weight (LMW) gelators, including modified amino acid derivatives, have been reported as viable alternatives to peptide-based materials. Herein, we report the encapsulation and release of proteins from supramolecular hydrogels composed of perfluorinated fluorenylmethyloxcarbonyl-modified phenylalanine (Fmoc-F5-Phe-DAP). Specifically, we demonstrate release of four model proteins (ribonuclease A (RNase A), trypsin inhibitor (TI), bovine serum albumin (BSA), and human immunoglobulin G (IgG)) from these hydrogels. The emergent viscoelastic properties of these materials are characterized, and the functional and time-dependent release of proteins from the hydrogels is demonstrated. In addition, it is shown that the properties of the aqueous solution used for hydrogel formulation have a significant influence on the in vitro release profiles, as a function of the isoelectric point and molecular weight of the protein payloads. These studies collectively validate that this class of supramolecular LMW hydrogel possesses the requisite properties for the sustained and localized release of protein therapeutics.