Proximity-enabled covalent binding of IL-2 to IL-2Rα selectively activates regulatory T cells and suppresses autoimmunity.

Interleukin-2 (IL-2) is a pleiotropic cytokine that orchestrates bidirectional immune responses via regulatory T cells (Tregs) and effector cells, leading to paradoxical consequences. Here, we report a strategy that exploited genetic code expansion-guided incorporation of the latent bioreactive artificial amino acid fluorosulfate-L-tyrosine (FSY) into IL-2 for proximity-enabled covalent binding to IL-2Rα to selectively promote Treg activation. We found that FSY-bearing IL-2 variants, such as L72-FSY, covalently bound to IL-2Rα via sulfur-fluoride exchange when in proximity, resulting in persistent recycling of IL-2 and selectively promoting the expansion of Tregs but not effector cells. Further assessment of L72-FSY-expanded Tregs demonstrated that L72-FSY maintained Tregs in a central memory phenotype without driving terminal differentiation, as demonstrated by simultaneously attenuated expression of lymphocyte activation gene-3 (LAG-3) and enhanced expression of programmed cell death protein-1 (PD-1). Subcutaneous administration of L72-FSY in murine models of pristane-induced lupus and graft-versus-host disease (GvHD) resulted in enhanced and sustained therapeutic efficacy compared with wild-type IL-2 treatment. The efficacy of L72-FSY was further improved by N-terminal PEGylation, which increased its circulatory retention for preferential and sustained effects. This proximity-enabled covalent binding strategy may accelerate the development of pleiotropic cytokines as a new class of immunomodulatory therapies.

CARs: a new approach for the treatment of autoimmune diseases.

The development of chimeric antigen receptor (CAR)-based therapeutic interventions represented a breakthrough in cancer treatment. Following the success of the CAR-T-cell strategy, this novel therapeutic approach has been applied to other diseases, including autoimmune diseases. Using CAR-T cells to deplete pathological immune cells (i.e., B cells, autoreactive B or T cells, and accessory antigen-presenting cells (APCs)) has resulted in favorable outcomes in diseases characterized by excessive autoantibody levels or hyperactive lymphocyte cell numbers. The importance of immunosuppressive regulatory T cells (Tregs) in restoring immune tolerance has been well established, and CAR-Tregs have shown promising therapeutic potential in treating autoimmune diseases. Moreover, prior experience from the cancer field has provided sufficient paradigms for understanding how to optimize the structure and function of CARs to improve their function, persistence, stability and safety. In this review, we describe the potential application of CAR-T cells and CAR-Tregs in the treatment of autoimmune diseases, and we summarize the currently available strategies of gene editing and synthetic biological tools that have improved the practical application of CAR-based therapies.

Self-fused concatenation of interferon with enhanced bioactivity, pharmacokinetics and antitumor efficacy.

We report an easy but universal protein modification approach, self-fused concatenation (SEC), to biosynthesize a set of interferon (IFN) concatemers with improved in vitro bioactivity, in vivo pharmacokinetics and therapeutic efficacy over the monomeric IFN, and the results can be positively enhanced by the concatenated number of self-fused proteins.

The role of vitamin D-synthesizing enzyme CYP27B1 in systemic lupus erythematosus.

BACKGROUND: To measure the expression of 1α-hydroxylase (CYP27B1) and serum 25(OH)D concentration in systemic lupus erythematosus (SLE) and to investigate the role of CYP27B1 in SLE. METHODS: Seventy-seven SLE patients and 35 healthy controls (HCs) were enrolled from September 2017 to January 2020. The study design is cross-sectional. mRNA expression of CYP27B1 in peripheral blood mononuclear cells (PBMCs) was measured by reverse-transcription quantitative PCR, the protein level of CYP27B1 was quantified by western blotting, and the serum level of 25(OH) D was determined by an enzyme-linked immunosorbent assay. RESULTS: The mRNA expression of CYP27B1 in PBMCs was significantly lower in SLE patients than in HCs (p < 0.001), and the protein quantification confirmed that CYP27B1 expression was lower in SLE patients than in HCs (p = 0.001). Among SLE patients, the prevalence of lupus nephritis was higher in a subgroup with lower CYP27B1 mRNA expression than in a subgroup with normal CYP27B1 mRNA expression (41.07% vs. 14.28%, p = 0.028). The mRNA expression of CYP27B1 negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (r = -0.331, p = 0.003). Serum 25(OH)D concentration was lower in SLE patients than in HCs (37.64 ± 19.89 vs. 50.58 ± 12.74 ng/mL, mean ± SD, p = 0.003). DISCUSSION: The expression of CYP27B1 in PBMCs may be related to SLE pathogenesis, disease activity, and nephritis.

Therapeutic potential of interleukin-2 in autoimmune diseases.

Autoimmune diseases are characterized by dysregulation and aberrant activation of cells in the immune system. Therefore, restoration of the immune balance represents a promising therapeutic target in autoimmune diseases. Interleukin-2 (IL-2) can promote the expansion and differentiation of different immune cell subsets dose-dependently. At high doses, IL-2 can promote the differentiation and expansion of effector and memory T cells, whereas at low doses, IL-2 can promote the differentiation, survival, and function of regulatory T (Treg) cells, a CD4+ T cell subset that is essential for the maintenance of immune homeostasis and immune tolerance. Therefore, IL-2 exerts immunostimulatory and immunosuppressive effects in autoimmune diseases. The immunoregulatory role of low-dose IL-2 has sparked excitement for the therapeutic exploration of modulating the IL-2-Treg axis in the context of autoimmune diseases. In this review, we discuss recent advances in the therapeutic potential of IL-2 or IL-2-derived molecules in the treatment of autoimmune diseases.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

Enhancement of bioactivity, thermal stability and tumor retention by self-fused concatenation of green fluorescent protein.

The widespread application of protein and peptide therapeutics is hampered by their poor stability, strong immunogenicity and short half-life. However, the existing protein modification technologies require the introduction of exogenous macromolecules, resulting in inevitable immunogenicity and decreased bioactivity. Herein, we reported an easy but universal protein modification approach, self-fused concatenation (SEC), to enhance the in vitro thermal stability and in vivo tumor retention of proteins. In this proof of concept study, we successfully obtained a set of green fluorescence protein (GFP) concatemers, monomer (GFP 1), dimer (GFP 2) and trimer (GFP 3) of GFP, and systematically studied the effects of SEC on the biological activity and stability of GFP. Notably, GFP concatemers displayed remarkable improvement in in vitro bioactivity and thermal stability over the monomeric GFP. In a murine tumor model, GFP 2 and GFP 3 exhibited significantly prolonged duration, with increases of 220- and 381-fold relative to GFP 1 in tumor retention 4 h after administration. Furthermore, the biological activity, thermal stability and tumor retention can be enhanced by the concatenated number of self-fused proteins. These findings demonstrate that SEC may be a promising alternative to design advanced protein and peptide therapeutics with enhanced pharmaceutic profiles.

Genetic Fusion of Transacting Activator of Transcription Peptide to Cyclized Green Fluorescence Protein Improves Stability, Intracellular Delivery, and Tumor Retention.

Therapeutic proteins such as enzymes, hormones, and cytokines suffer from poor stability, inefficient cellular penetration, and rapid clearance from circulation. Conjugation with polymers (such as poly(ethylene glycol)) and fusion with long-acting proteins (such as albumin and Fc fragments) have been utilized to partially address the delivery issues, but these strategies require the introduction of new macromolecular substances, resulting in potential immunogenicity and toxicity. Herein, we report an easy strategy to increase the intracellular delivery efficiency and stability of proteins by combining of sortase-mediated protein cyclization and cell-penetrating peptide (CPP)-mediated intracellular delivery. We, for the first time, genetically constructed a green fluorescence protein (GFP) fused with a CPP, a transacting activator of transcription (TAT) peptide, at its C-terminus for intracellular internalization, and two sortase recognition sequences, pentaglycine and LPETG, at its N- and C-termini for cyclization. Notably, the cyclized GFP-TAT (cGFP-TAT) not only highly retained the photophysical properties of the protein but also significantly improved the in vitro stability compared with the native linear GFP (lGFP) and linear TAT peptide-fused GFP (lGFP-TAT).Moreover, cGFP-TAT showed better cellular internalization ability compared with lGFP. In C26 tumor-inoculated mice, cGFP-TAT exhibited enhanced in vivo tumor retention, with increases of 7.79- and 6.52-fold relative to lGFP and lGFP-TAT in tumor retention 3 h after intratumor administration. This proof-of-concept study has provided an easy strategy to increase the in vitro stability, intracellular delivery efficiency, and in vivo tumor retention of GFP, which would be applicable to numerous therapeutic proteins and peptides for clinical practice.

In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.

OBJECTIVES: Autoreactive B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA), and B cell-depleting therapies using an antibodies, such as rituximab, have been suggested to be effective in RA treatment. However, transient B cell depletion with rituximab is associated with significant safety challenges related to global suppression of the immune system and thus increases the risks of infection and cancer development. To address selective and persistent issues associated with RA therapy, we developed a customised therapeutic strategy employing universal antifluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) combined with FITC-labelled antigenic peptide epitopes to eliminate autoreactive B cell subsets recognising these antigens in RA. METHODS: For a proof-of-concept study, four citrullinated peptide epitopes derived from citrullinated autoantigens, namely, citrullinated vimentin, citrullinated type II collagen, citrullinated fibrinogen and tenascin-C, and a cyclocitrulline peptide-1 were selected as ligands for targeting autoreactive B cells; Engineered T cells expressing a fixed anti-FITC CAR were constructed and applied as a universal CAR-T cell system to specifically eliminate these protein-specific autoreactive B cells via recognition of the aforementioned FITC-labelled autoantigenic peptide epitopes. RESULTS: We demonstrated that anti-FITC CAR-T cells could be specifically redirected and kill hybridoma cells generated by immunisation with antigenic peptides, and autoreactive B cell subsets from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes. Additionally, the cytotoxicity of the CAR-T cells was dependent on the presence of the peptides and occurred in a dose-dependent manner. CONCLUSIONS: The approach described here provides a direction for precise, customised approaches to treat RA and can likely be applied to other systemic autoimmune diseases.

[Increased expression of LC3 in peripheral blood mononuclear cells is related to disease status in patients with rheumatoid arthritis].

Objective To detect the mRNA and protein expression of microtubule-associated protein 1 light chain 3 (LC3) in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA), and to investigate its relationship with RA. Methods Twenty-two patients with RA and 16 healthy subjects with matching gender and age as controls were included in the study. PBMCs were isolated by density gradient centrifugation. The level of LC3 mRNA in PBMCs was detected by real-time fluorescent quantitative PCR. The protein level of LC3 in PBMCs was detected by Western blot analysis. The expression of LC3 protein in PBMCs was detected by immunofluorescence staining. Pearson analysis was used to analyze the correlation between LC3 expression and clinical parameters of RA patients. Results Compared with the normal control group, the levels of LC3 mRNA and protein in PBMCs of RA patients went up significantly, and the expression of LC3 significantly increased in PBMCs. The mRNA expression level of LC3 was obviously positively correlated with erythrocyte sedimentation rate (ESR, r=0.7480), 28 joint disease activity (DAS28, r=0.5016), C-reactive protein (CRP, r=0.6518), and rheumatoid factor (RF, r=0.7232). Conclusion The expression of LC3 is up-regulated in RA patients and is associated with ESR, DAS28, CRP and RF.