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We show that our machine learning models predict Met and Trp photooxidation likelihood with 0.926 and 0.860 area under the curve (AUC), respectively, and Met photooxidation rate with a correlation coefficient (Q2) of 0.511 and root-mean-square error (RMSE) of 10.9%. We further identify important physical, chemical, and formulation parameters that influence photooxidation. Improvement of biopharmaceutical liability predictions will result in better, more stable drugs, increasing development throughput, product quality, and likelihood of clinical success.Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGFFlt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect. CB T cells are readily available in CB banks and show low toxicity, high proliferation rates, and increased anti-leukemic effect upon transfer. However, the combination of anti-tumor gene modification and preservation of advantageous immunological traits of CB T cells represent major challenges for the harmonized production of T cell therapy products. In this manuscript, we optimized a protocol for expansion and lentiviral vector (LV) transduction of CB CD8+ T cells, achieving a transduction efficiency up to 83%. Timing of LV treatment, selection of culture media, and the use of different promoters were optimized in the transduction protocol. LentiBOOST was confirmed as a non-toxic transduction enhancer of CB CD8+ T cells, with minor effects on the proliferation capacity and cell viability of the T cells. Positively, the use of LentiBOOST does not affect the functionality of the cells, in the context of tumor cell recognition. Finally, CB CD8+ T cells were more amenable to LV transduction than peripheral blood (PB) CD8+ T cells and maintained a more naive phenotype. In conclusion, we show an efficient method to genetically modify CB CD8+ T cells using LV, which is especially useful for off-the-shelf adoptive cell therapy products for cancer treatment.Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. α-cyano-4-hydroxycinnamic mw The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial.The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo. Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy.Antigen-specific lung-resident memory T cells (TRMs) constitute the first line of defense that mediates rapid protection against respiratory pathogens and inspires novel vaccine designs against infectious pandemic threats, yet effective means of inducing TRMs, particularly via non-viral vectors, remain challenging. Here, we demonstrate safe and potent induction of lung-resident TRMs using a biodegradable polymeric nanoshell that co-encapsulates antigenic peptides and TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) in a virus-mimicking structure. Through subcutaneous priming and intranasal boosting, the combinatorial nanoshell vaccine elicits prominent lung-resident CD4+ and CD8+ T cells that surprisingly show better durability than live viral infections. In particular, nanoshells containing CpG-ODN and a pair of conserved class I and II major histocompatibility complex-restricted influenza nucleoprotein-derived antigenic peptides are demonstrated to induce near-sterilizing immunity against lethal infections with influenza A viruses of different strains and subtypes in mice, resulting in rapid elimination of replicating viruses. We further examine the pulmonary transport dynamic and optimal composition of the nanoshell vaccine conducive for robust TRM induction as well as the benefit of subcutaneous priming on TRM replenishment. The study presents a practical vaccination strategy for inducing protective TRM-mediated immunity, offering a compelling platform and critical insights in the ongoing quest toward a broadly protective vaccine against universal influenza as well as other respiratory pathogens.While virus-specific antibodies are broadly recognized as correlates of protection, virus-specific T cells are important for direct clearance of infected cells. Failure to generate hepatitis B virus (HBV)-specific antibodies is well-known in patients with end-stage renal disease. However, whether and to what extent HBV-specific cellular immunity is altered in this population and how it influences humoral immunity is not clear. To address it, we analyzed HBV-reactive T cells and antibodies in hemodialysis patients post vaccination. 29 hemodialysis patients and 10 healthy controls were enrolled in a cross-sectional study. Using multiparameter flow cytometry, HBV-reactive T cells were analyzed and functionally dissected based on granzyme B, interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and IL-4 expression. Importantly, HBV-reactive CD4+ T cells were detected not only in all patients with sufficient titers but also in 70% of non-responders. Furthermore, a correlation between the magnitude of HBV-reactive CD4+ T cells and post-vaccination titers was observed. In summary, our data showed that HBV-reactive polyfunctional T cells were present in the majority of hemodialysis patients even if humoral immunity failed. Further studies are required to confirm their in vivo antiviral capacity. The ability to induce vaccine-reactive T cells paves new ways for improved vaccination and therapy protocols.Eosinophilic gastroenteritis is a rare entity, and although usually idiopathic, it may arise from gastrointestinal infections. We report a case of a 33-year-old woman from Vietnam presenting with acute abdominal pain, new-onset eosinophilic ascites, peripheral eosinophilia, and a positive Toxocara antibody. The patient had no recent international travel, known animal host contact, or other toxocariasis risk factors. She was treated with ivermectin and mebendazole with complete resolution of symptoms. This case emphasizes the consideration of a broad differential for eosinophilic ascites, including atypical presentations of infectious pathogens.
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