Search

Self-association governs the viscosity and solubility of therapeutic antibodies in high-concentration formulations used for subcutaneous delivery, yet it is difficult to reliably identify candidates with low self-association during antibody discovery and early-stage optimization. Here, we report a high-throughput protein engineering method for rapidly identifying antibody candidates with both low self-association and high affinity. We find that conjugating quantum dots to IgGs that strongly self-associate (pH 7.4, PBS), such as lenzilumab and bococizumab, results in immunoconjugates that are highly sensitive for detecting other high self-association antibodies. Moreover, these conjugates can be used to rapidly enrich yeast-displayed bococizumab sub-libraries for variants with low levels of immunoconjugate binding. Deep sequencing and machine learning analysis of the enriched bococizumab libraries, along with similar library analysis for antibody affinity, enabled identification of extremely rare variants with co-optimized levels of low self-association and high affinity. This analysis revealed that co-optimizing bococizumab is difficult because most high-affinity variants possess positively charged variable domains and most low self-association variants possess negatively charged variable domains. Moreover, negatively charged mutations in the heavy chain CDR2 of bococizumab, adjacent to its paratope, were effective at reducing self-association without reducing affinity. Interestingly, most of the bococizumab variants with reduced self-association also displayed improved folding stability and reduced nonspecific binding, revealing that this approach may be particularly useful for identifying antibody candidates with attractive combinations of drug-like properties.

The purpose of this study is to evaluate the efficacy and safety of vaginal fractional CO2 laser therapy for female sexual dysfunction (FSD). A total of 84 women at high risk of sexual dysfunction were randomly divided into two groups. Women in the laser group received vaginal fractional CO2 laser therapy. Others in the Kegel group were advised to participate in Kegel exercise training. Sexual distress and sexual function were evaluated by using the Female Sexual Distress Scale-Revised (FSDS-R) and the Chinese version Female Sexual Function Index (CVFSFI), respectively. Adverse events were recorded during the 12-month follow-up. At the end of the 6th and 12th months, the lubrication scores of the CVFSFI in the laser group (4.55±0.05, 4.58±0.09) were significantly higher than those in the Kegel group (4.19±0.15, 4.20±0.14) (P

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-(13)C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-(13)C]glucose to [(13)C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein–lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Biologics such as peptides and proteins possess a number of attractive attributes that make them particularly valuable as therapeutics, including their high activity, high specificity, and low toxicity. However, one of the key challenges associated with this class of drugs is their propensity to aggregate. Given the safety and immunogenicity concerns related to polypeptide aggregates, it is particularly important to sensitively detect aggregates in therapeutic drug formulations as part of the quality control process. Here, we report the development of conformation-specific antibodies that recognize polypeptide aggregates composed of a GLP-1 receptor agonist (liraglutide) and their integration into a sensitive immunoassay for detecting liraglutide amyloid fibrils. We sorted single-chain antibody libraries against liraglutide fibrils using yeast surface display and magnetic-activated cell sorting, and identified several antibodies with high conformational specificity. Interestingly, these antibodies cross-react with amyloid fibrils formed by several other polypeptides, revealing that they recognize molecular features common to different types of fibrils. Moreover, we find that our immunoassay using these antibodies is >50-fold more sensitive than the conventional method for detecting liraglutide aggregation (Thioflavin T fluorescence). We expect that our systematic approach for generating a sensitive, aggregate-specific immunoassay can be readily extended to other biologics to improve the quality and safety of formulated drug products.

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.

The purpose of this study is to evaluate the efficacy and safety of vaginal fractional CO

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CLOX, has been implicated in the cellular signaling events that initiate apoptosis. CLOX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ12-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CLOX, and treatment of cells with H2O2 increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects, and in humans, this results in the life-threatening disorder Barth syndrome. Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL-specific phospholipase, Cld1, that can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin-deficient cells are caused by the decreased CL/MLCL ratio and not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the heme activator protein transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1-mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiration.

Introduction Recent years have seen continuous reports about the successful reconstruction of numerous organs with the application of tissue‐engineering techniques. Thus, we assess the outcomes for vagina reconstruction using tissue‐engineered biological material, which we suggested previously as an ideal graft for vaginoplasty. Aim To evaluate the anatomic and sexual outcomes in patients undergoing vaginoplasty using tissue‐engineered biomaterial mesh. Methods This prospective study included 53 patients with Mayer‐Rokitansky‐Kuster‐Hauser syndrome admitted to our hospital. Patients underwent vaginoplasty with tissue‐engineered biological material (acellular dermal matrix). Postoperatively, a silicone vaginal dilator (length: 10 cm, diameter: 3.5 cm) was advised to be used for the first 3–6 months to prevent contraction of the neovagina. Follow‐up was performed at 4 weeks, 12 weeks, 12 months, and then annually. Twenty‐four age‐matched women who underwent health examinations during the same time period were selected as a health control group and answered Female Sexual Function Index (FSFI) questionnaires for the purpose of comparing sexuality. Main Outcome Measures Anatomic success was defined by a vaginal length ≥8 cm and a width allowing the easy introduction of two fingers. Sexual outcomes were assessed at the 12‐month follow‐up according to body image perception and FSFI questionnaires validated for the Chinese‐speaking population. Results No severe intra‐operative complications occurred. No graft‐related infection, rejection, or detachment was recorded. The cost for tissue‐engineered biomaterial graft was $1,900 (¥12,000) per person. Postoperatively, granulomatous polyps occurred in 6/53 patients (11.3%) at the vaginal vault and were removed in an outpatient clinic. During a mean follow‐up of 21.1 months, the anatomic success rate was 100%, and all of the patients were satisfied with their body image. Postoperatively, 42 patients were followed up for more than 1 year, and 32 of them were sexually active. Among the 24/32 patients (75%) who answered the FSFI questionnaire, the mean total FSFI score was 26.7 ± 3.5, which was similar to that of the control group (25.6 ± 7.4, P = 0.46). The similarity was also observed in six separate domains of the functional aspect of female sexuality. Conclusions Vaginoplasty with tissue‐engineered biomaterial graft is a safe, effective, minimally invasive cosmetic procedure that provides near normal sexual function for patients with vaginal aplasia.

It has been demonstrated that numerous microRNAs (miRNAs) have potent tumor-suppressing effects on a variety of cancers, implicating a possible application of miRNA in tumor therapy. Oncolytic adenovirus is a suitable vector to deliver tumor suppressor genes for treatment of cancers. However, it remains unknown whether co-expression of tumor suppressor genes and miRNAs can contribute to a more potent antitumor capacity within an oncolytic adenovirus delivery system. In this study, we found that expression of miRNA-34a was reduced in hepatocellular carcinoma (HCC), and the reduced expression of miRNA-34a was associated with worse outcome of HCC patients. Thus, we developed an oncolytic adenoviral vector, AdCN205, to co-express miRNA-34a and IL-24 driven by an adenovirus endogenous E3 promoter in HCC cells. High levels of miRNA-34a and IL-24 expression were detected in AdCN205-IL-24-miR-34a-infected HCC cells. AdCN205-IL-24-miR-34a significantly induced dramatic antitumor activity, as compared with that induced by AdCN205-IL-24 or AdCN205-miR-34a alone. Transfer of miRNA-34a into HCC cells inhibited the expression of its target genes, Bcl-2 and SIRT1. Treatment of established xenograft HCC tumors with AdCN205-IL-24-miR-34a in a mouse model resulted in complete tumor regression without recurrence. Taken together, our data provide a promising and reasonable delivery strategy of double-aimed cancer therapy, in which miRNAs and tumor-suppressing genes are used simultaneously.

Purpose: Recent studies have indicated that short hairpin RNA (shRNA) driven by RNA polymerase (Pol) II promoters can be transcribed into precursor mRNAs together with transgenes. It remains unclear, however, whether coexpression of shRNA and transgene from a single promoter is feasible for cancer therapy. Experimental Design: In this study, we generated novel adenoviral vectors that permitted coexpression of shRNA against cyclooxygenase-2 (COX-2) and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapeutic gene from a cytomegalovirus promoter to evaluate whether silencing of COX-2 could increase the sensitivity of hepatocellular carcinoma to TRAIL. Results: Our data showed that adenovirus vector Ad-TM, in which the shRNA was inserted into the 3′ untranslated region of the TRAIL gene, not only significantly suppressed COX-2 expression, but also expressed a high level of TRAIL. Moreover, infection with Ad-TM resulted in significant cytotoxicity in hepatocellular carcinoma cell lines. In contrast, it had no effect on normal liver cell line. Impressively, treatment of the established hepatocellular carcinoma tumors with Ad-TM resulted in complete tumor regression. This potent antitumor activity induced by Ad-TM was due to strong inhibition of COX-2 and high expression of TRAIL. Furthermore, using the shRNA and transgene coexpression adenovirus system, we showed that silencing of COX-2 increased the sensitivity of hepatocellular carcinoma to TRAIL through inhibition of Bcl-2 and Bcl-w. Conclusion: This study indicated that adenovirus carrying shRNA and transgene expressed from a single promoter represented a potent approach for cancer therapy. Clin Cancer Res; 16(14); 3696–705. ©2010 AACR.

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects and, in humans, results in the life-threatening disorder Barth syndrome (BTHS). Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL-specific phospholipase, Cld1, which can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin-deficient cells is caused by the decreased CL/MLCL ratio, not by a deficiency in unsaturated CL. CLD1 expression is inc...

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in diverse cancers, which contributes to the proliferation and survival of cancer cells by upregulating apoptosis inhibitors and cell cycle regulators. Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator of STAT pathways and is frequently silenced in many types of cancers. In this study, we used oncolytic adenoviral vector to deliver SOCS1 gene (AdCN305-SOCS1) to treat hepatocellular carcinoma (HCC). Our data showed that SOCS1 was downregulated in HCC cells by hypermethylation. AdCN305-SOCS1 was found selectively replicated, which led to SOCS1 overexpression in HCC cells. Infection of HCC cells with AdCN305-SOCS1 resulted in inhibition of STAT3 phosphorylation and downregulation of survivin, cyclin D1, Bcl-xL and C-myc. AdCN305-SOCS1 exhibited strong cytotoxicity to HCC cells by inducing apoptosis in vitro and in vivo. This study suggests that transfer of SOCS1 by an oncolytic adenovirus may be a potent antitumor approach for cancer therapy.