A Hydroxylamine-Mediated Amidination of Lysine Residues That Retains the Protein's Positive Charge.

Lysine-specific peptide and protein modification strategies are widely used to study charge-related functions and applications. However, these strategies often result in the loss of the positive charge on lysine, significantly impacting the charge-related properties of proteins. Herein, we report a strategy to preserve the positive charge and selectively convert amines in lysine side chains to amidines using nitriles and hydroxylamine under aqueous conditions. Various unprotected peptides and proteins were successfully modified with a high conversion rate. Moreover, the reactive amidine moiety and derived modification site enable subsequent secondary modifications. Notably, positive charges were retained during the modification. Therefore, positive charge-related protein properties, such as liquid-liquid phase separation behaviour of α-synuclein, were not affected. This strategy was subsequently applied to a lysine rich protein to develop an amidine-containing coacervate DNA complex with outstanding mechanical properties. Overall, our innovative strategy provides a new avenue to explore the characteristics of positively charged proteins.

Regulation of Immune Activation by Optical Control of TLR1/2 Heterodimerization.

The activation of toll-like receptors (TLRs) plays important roles in the immune response. The ability to control the activities of TLRs could be usable as a switch for immune response. Here we have rationally designed and synthesized a photoswitchable Pam3 CSK4 derivative-P10-to control the activation of TLR1/2. The ground-state trans-P10 was able to stimulate and activate antigen-presenting cells (APCs) by promoting TLR1/2 heterodimerization. However, cis-P10, derived from UV irradiation of trans-P10, reduced the activities of APCs by impeding the TLR1/2 heterodimerization. In the absence of UV radiation, the cis-P10 slowly returned to its ground trans state, restoring the activities of the APCs stimulation. Our results indicated that optical control of TLR1/2 heterodimerization mediated by the photoswitchable P10 offers the potential to regulate immune activation and inflammation.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.

Self-Assembled Nano-Immunostimulant for Synergistic Immune Activation.

Immunotherapy has become one of the most promising therapies for the treatment of diseases. Synthetic immunostimulants and nanomaterial immunostimulant systems are indispensable for the activation of the immune system in cancer immunotherapy. Herein, a strategy for preparing self-assembled nano-immunostimulants (SANIs) for synergistic immune activation is reported. Three immunostimulants self-assemble into nanoparticles through electrostatic interactions. SANIs showed strong synergistic immunostimulation in macrophages. SANIs could also induce a strong antitumor immune response to inhibit tumor growth in mice and act as an efficient adjuvant of antitumor vaccines. Therefore, SANIs may be generally applied in cancer immunotherapy. This novel SANI strategy provides a new way for the development of both immunostimulants and -suppressants.

Glycopeptide Nanoconjugates Based on Multilayer Self-Assembly as an Antitumor Vaccine.

Antitumor vaccine, which is promising for tumor therapy, has been extensively studied. Some encouraging results of chemically synthetic vaccine designs based on the tumor-associated antigen mucin 1 have been achieved. However, some shortcomings such as low efficiency and difficult purification restrict their clinical application. To overcome these difficulties, we designed a novel antitumor vaccine of glycopeptide nanoconjugates based on the multilayer self-assembly through the interaction of positive and negative charges. This vaccine formed the spherical structure and effectively activated the macrophage in vitro. Besides, it also induced high titer of antibodies against mucin 1 glycopeptide. The induced antibodies could highly bind to the tumor cells and effectively kill them by activation of the complement dependent cytotoxicity complex. This novel strategy provides a new way for the development of simple and effective antitumor vaccine.

Covalent bond or noncovalent bond: a supramolecular strategy for the construction of chemically synthesized vaccines.

A novel noncovalent strategy to construct chemically synthesized vaccines has been designed to trigger a robust immune response and to dramatically improve the efficiency of vaccine preparation. Glycosylated MUC1 tripartite vaccines were constructed through host-guest interactions with cucurbit[8]uril. These vaccines elicited high levels of IgG antibodies that were recognized by transformed cells and induced the secretion of cytokines. The antisera also mediated complement-dependent cytotoxicity. This noncovalent strategy with good suitability, scalability, and feasibility can be applied as a universal strategy for the construction of chemically synthesized vaccines.

Uncovering the pathological functions of Ser404 phosphorylation by semisynthesis of a phosphorylated TDP-43 prion-like domain.

Herein, we have successfully semi-synthesized a TDP-43 prion-like domain with Ser404 phosphorylation. We have demonstrated that Ser404 phosphorylation could accelerate the amyloid aggregation of the TDP-43 prion-like domain and aggravate its cytotoxicity.

Automated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy.

High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases.

Targeting STING with cyclic di-GMP greatly augmented immune responses of glycopeptide cancer vaccines.

Cyclic di-GMP (CDG) was applied to MUC1 glycopeptide-based cancer vaccines with physical mixing and built-in (at 2'-OH of CDG) strategies for activating the STING pathway. CDG in both strategies behaved as a potent immunostimulant and contributed to high titers of IgG antibodies and the expression of multiple cytokines.

Selective inhibition of cancer cells by enzyme-induced gain of function of phosphorylated melittin analogues.

The selective killing of cancer cells and the avoidance of drug resistance are still difficult challenges in cancer therapy. Here, we report a new strategy that uses enzyme-induced gain of function (EIGF) to regulate the structure and function of phosphorylated melittin analogues (MelAs). Original MelAs have the capacity to disrupt plasma membranes and induce cell death without selectivity. However, phosphorylation of Thr23 on one of the MelAs (MelA2-P) efficiently ameliorated the membrane lysis potency as well as the cytotoxicity for normal mammalian cells. After treatment with alkaline phosphatase (ALP), which is more active in cancer cells than normal cells, MelA2-P restored the pore-forming function around the cancer cells and induced cancer cell death selectively. This mechanism was independent of the receptor proteins and the cell uptake process, which may partially bypass the development of drug resistance in cancer cells.

Prophylactic Vaccine Based on Pyroglutamate-3 Amyloid ß Generates Strong Antibody Response and Rescues Cognitive Decline in Alzheimer's Disease Model Mice.

Clearance of amyloid ß (Aß) by immunotherapy is one of the fancy methods to treat Alzheimer's disease (AD). However, the failure of some clinical trials suggested that there may be something ignored in the past development of immunotherapy. Pyroglutamate-3 Aß (AßpE3-X), which was found to be abundant in the patients' brain, has attracted much attention after the report that AßpE3-42 could serve as a template to exacerbate the aggregation of Aß. In addition, AßpE3-X could not be recognized by the antibodies targeting the N-terminus of Aß, suggesting that AßpE3-X maybe the ignored one. Indeed, passive immunization targeting AßpE3-X has shown some beneficial results, while active immunotherapy has not been extensively studied. In the present study, we designed and synthesized a novel peptide vaccine targeting AßpE3-X, which contains AßpE3-15 as B cell epitope and P2 as T cell epitope. We showed that this vaccine could induce strong antibody response to AßpE3-X. We also showed that prophylactic immunization of AD model mice with our vaccine could reduce Aß plaques and rescue cognitive decline. This new kind of Aß vaccine will open up new directions for AD immunotherapy.

Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation.

Tau, an important pathological protein of Alzheimer's disease (AD), can mediate the toxicity of amyloid ß (Aß). Thus, reduction of Tau with chemical molecules may offer a novel strategy for treating AD. Here, we designed and synthesized a series of multifunctional molecules that contained Tau-recognition moieties and E3 ligase-binding moieties to enhance Tau degradation. Among these molecules, TH006 had the highest activity of inducing Tau degradation by increasing its poly-ubiquitination. The decrement in Tau induced by TH006 could decrease the cytotoxicity caused by Aß. Furthermore, TH006 could regulate the Tau level in the brain of an AD mouse model. Therefore, partial reduction of Tau with such multifunctional peptides may open up a novel therapeutic strategy for AD treatment.

Strategy for Designing a Synthetic Tumor Vaccine: Multi-Component, Multivalency and Antigen Modification.

Synthetic tumor vaccines have been proven to be promising for cancer immunotherapy. However, the limitation of the specificity and efficiency of the synthetic tumor vaccines need further improvements. To overcome these difficulties, additional tumor-associated targets need to be identified, and optimized structural designs of vaccines need to be elaborated. In this review, we summarized the main strategies pursued in the design of synthetic tumor vaccines, such as multi-component, multivalency, antigen modification and other possible ways to improve the efficiency of synthetic tumor vaccines.