Fabrication of Interleukin-4 Encapsulated Bioactive Microdroplets for Regulating Inflammation and Promoting Osteogenesis.

Background: Despite the inherent regenerative ability of bone, large bone defect regeneration remains a major clinical challenge for orthopedic surgery. Therapeutic strategies medicated by M2 phenotypic macrophages or M2 macrophage inducer have been widely used to promote tissue remodeling. In this study, ultrasound-responsive bioactive microdroplets (MDs) encapsulated with bioactive molecule interleukin-4 (IL4, hereafter designated MDs-IL4) were fabricated to regulate macrophage polarization and potentiate the osteogenic differentiation of human mesenchymal stem cells (hBMSCs). Materials and Methods: The MTT assay, live and dead staining, and phalloidin/DAPI dual staining were used to evaluate biocompatibility in vitro. H&E staining was used to evaluate biocompatibility in vivo. Inflammatory macrophages were further induced via lipopolysaccharide (LPS) stimulation to mimic the pro-inflammatory condition. The immunoregulatory role of the MDs-IL4 was tested via macrophage phenotypic marker gene expression, pro-inflammatory cytokine level, cell morphological analysis, and immunofluorescence staining, etc. The immune-osteogenic response of hBMSCs via macrophages and hBMSCs interactions was further investigated in vitro. Results: The bioactive MDs-IL4 scaffold showed good cytocompatibility in RAW 264.7 macrophages and hBMSCs. The results confirmed that the bioactive MDs-IL4 scaffold could reduce inflammatory phenotypic macrophages, as evidenced by changing in morphological features, reduction in pro-inflammatory marker gene expression, increase of M2 phenotypic marker genes, and inhibition of pro-inflammatory cytokine secretion. Additionally, our results indicate that the bioactive MDs-IL4 could significantly enhance the osteogenic differentiation of hBMSCs via its potential immunomodulatory properties. Conclusion: Our results demonstrate that the bioactive MDs-IL4 scaffold could be used as novel carrier system for other pro-osteogenic molecules, thus having potential applications in bone tissue regeneration.

A novel adoptive synthetic TCR and antigen receptor (STAR) T-Cell therapy for B-Cell acute lymphoblastic leukemia.

We developed a T-cell-receptor (TCR) complex-based chimeric antigen receptor (CAR) named Synthetic TCR and Antigen Receptor (STAR). Here, we report pre-clinical and phase I clinical trial data (NCT03953599) of this T-cell therapy for refractory and relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) patients. STAR consists of two protein modules each containing an antibody light or heavy chain variable region and TCR α or ß chain constant region fused to the co-stimulatory domain of OX40. T-cells were transduced with a STAR-OX40 lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR/STAR-OX40 T cell antitumor activity. Eighteen patients with R/R B-ALL were enrolled into the clinical trial. In a xenograft mouse model, STAR-T-cells exhibited superior tumor-specific cytotoxicity compared with conventional CAR-T cells. Incorporating OX40 into STAR further improved the proliferation and persistence of tumor-targeting T-cells. In our clinical trial, 100% of patients achieved complete remission 4 weeks post-STAR-OX40 T-cell infusion and 16/18 (88.9%) patients pursued consolidative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Twelve of 16 patients (75%) remained leukemia-free after a median follow-up of 545 (433-665) days. The two patients without consolidative allo-HSCT relapsed on Day 58 and Day 186. Mild cytokine release syndrome occurred in 10/18 (55.6%) patients, and 2 patients experienced grade III neurotoxicity. Our preclinical studies demonstrate super anti-tumor potency of STAR-OX40 T-cells compared with conventional CAR-T cells. The first-in-human clinical trial shows that STAR-OX40 T-cells are tolerable and an effective therapeutic platform for treating R/R B-ALL.

Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors.

Chimeric antigen receptor T (CAR-T) cell therapies have demonstrated high response rate and durable disease control for the treatment of B cell malignancies. However, in the case of solid tumors, CAR-T cells have shown limited efficacy, which is partially attributed to intrinsic defects in CAR signaling. Here, we construct a double-chain chimeric receptor, termed as synthetic T cell receptor (TCR) and antigen receptor (STAR), which incorporates antigen-recognition domain of antibody and constant regions of TCR that engage endogenous CD3 signaling machinery. Under antigen-free conditions, STAR does not trigger tonic signaling, which has been reported to cause exhaustion of traditional CAR-T cells. Upon antigen stimulation, STAR mediates strong and sensitive TCR-like signaling, and STAR-T cells exhibit less susceptibility to dysfunction and better proliferation than traditional 28zCAR-T cells. In addition, STAR-T cells show higher antigen sensitivity than CAR-T cells, which holds potential to reduce the risk of antigen loss-induced tumor relapse in clinical use. In multiple solid tumor models, STAR-T cells prominently outperformed BBzCAR-T cells and generated better or equipotent antitumor effects to 28zCAR-T cells without causing notable toxicity. With these favorable features endowed by native TCR-like signaling, STAR-T cells may provide clinical benefit in treating refractory solid tumors.