Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation.

BACKGROUND: The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. RESULTS: In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. CONCLUSIONS: The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

In vivo study of newly developed albumin-conjugated urate oxidase for gout treatment.

BACKGROUND: Exogenously providing engineered Uox with enhanced half-life is one of the important urate-lowering treatments for gout. The potential of PAT101, a recombinant human albumin (rHA)-conjugated variant, was evaluated and compared as a novel gout treatment through various in vivo studies with PAT101 and competing drugs. METHODS: PAT101 was produced by site-specific conjugation of rHA and Aspergillus flavus Uox (AfUox-rHA) through clickable non-natural amino acid (frTet) and Inverse electron demand Diels-Alder (IEDDA) reaction. In vivo pharmacokinetics, efficacy tests and in vitro immunogenetic assay were performed after single or multiple doses of PAT101 and its competitors in BALB/c mice, transgenic (TG) mice, Sprague-Dawley (SD) rats, and non-human primate (NHP). RESULTS: The half-life of PAT101 in single-dose treated TG mice was more than doubled compared to pegloticase. In SD rats with 4 weeks of repeated administration of rasburicase, only 24% of Uox activity remained, whereas in PAT101, it was maintained by 86%. In the Uox KO model, the survival rate of PAT101 was comparable to that of pegloticase. In addition, human PBMC-based CD4+/CD8+ T-cell activation analysis demonstrated that PAT101 has a lower immune response compared to the original drug, rasburicase. CONCLUSION: All results suggest that this rHA-conjugated AfUox, PAT101, can be provided as a reliable source of Uox for gout treatment.

Krill Oil Inhibits Cholesterol Synthesis and Stimulated Cholesterol Excretion in Hypercholesterolemic Rats.

The present study aimed to investigate the antihypercholesterolemic effects of krill oil supplementation in high-cholesterol diet-induced hypercholesterolemic rats, and the mechanisms underlying these effects. Rats were divided into five groups: normal control, control (high-cholesterol diet), krill oil 100 mg/kg b.w. (high-cholesterol diet with Krill oil 100 mg/kg b.w.), and krill oil 200 mg/kg b.w. (high-cholesterol diet with Krill oil 200 mg/kg b.w.). After 12 weeks, the rats were sacrificed to observe the effects of krill oil on cholesterol synthesis and excretion. We found that krill oil supplementation suppressed total triglycerides, total cholesterol, and LDL-cholesterol levels, as well as HMG-CoA reductase activity. It stimulated AMPK phosphorylation, LDL receptor and ACAT2 expression in the liver, and the fecal output of cholesterol. Furthermore, it decreased the levels of P-selectin, sVCAM-1, and NO, as well as aortic wall thickness, demonstrating its role in the prevention of atherosclerosis. Thus, we suggest that krill oil supplementation can reduce LDL-cholesterol levels in the blood during hypercholesterolemia by stimulating the uptake of LDL-cholesterol into tissue and cholesterol excretion, as well as inhibition of cholesterol synthesis.

Minimally invasive adenomyomectomy via a laparoscopic-assisted approach compared to a laparoscopic or laparotomic approach.

OBJECTIVE: The present study assessed the safety and benefits of laparoscopic-assisted adenomyomectomy compared to laparoscopic or laparotomic adenomyomectomy. MATERIALS AND METHODS: This study was a retrospective comparative study. A total of 277 patients underwent adenomyomectomy between January 2016 and January 2019 at the Department of Obstetrics and Gynaecology, Ulsan University Hospital, including 25 with laparoscopic-assisted adenomyomectomy, 82 with laparoscopic adenomyomectomy, and 170 with laparotomic adenomyomectomy. Laparoscopic-assisted adenomyomectomy consisted of a laparoscopic uterine artery procedure to reduce blood loss and a minimal incisional for laparotomic adenomyomectomy. An additional laparoscopic surgery was performed for possible pelvic pathology. RESULTS: Data on patient demographics, surgical indications, operative times, estimated blood loss (EBL), short-term complications, and postoperative hospital stays were compared. The laparoscopic-assisted surgery (LAS) and laparotomic groups were comparable in average EBL (208.0 ± 128.8 vs. 193.6 ± 193.0 ml, p = 0.11), weight of removed mass (85.5 ± 71.7 vs. 108.2 ± 91.9 g, p = 0.39), and postoperative hospital days (HDs) (4.5 ± 1.0 vs. 4.7 ± 0.8 days, p = 0.27). These values were lower in the laparoscopic group (EBL 119.5 ± 79.6 ml, mass weight 39.3 ± 25.9 g, HD 3.6 ± 0.8 days). Additional procedures, including myomectomy and combined severe endometriosis surgery, were more frequently performed in the LAS group than the laparotomic group. The mean operating time was longer in the LAS group (179.8 ± 36.6 min) than the other groups (laparoscopy 99.9 ± 40.6 min, p < 0.00; laparotomy 133.0 ± 41.1 min, p < 0.00). The three groups did not differ significantly in transfusion rates, hemoglobin changes, or perioperative complications. However, febrile morbidity was lower in the laparoscopic group than the LAS and laparotomic groups. CONCLUSION: LAS adenomyomectomy allows for maximal debulking of adenomyosis via extracorporeal and intracorporeal procedures while retaining the advantages of the laparoscopic approach. Additional pelvic surgery for benign uterine and adnexal pathology may easily be performed with this approach.

The Diagnostic Accuracy and Postoperative Outcomes of Cervical Cancer Patients for MR-invisible or MR-visible Diagnosis of Combined T2- and Diffusion-weighted 3T MRI Using the External Phased-array Receiver.

BACKGROUND/AIM: This study aimed to determine the diagnostic accuracy and postoperative outcomes of early-stage cervical cancer patients [2009 FIGO stages IA2-IB1 (<2 cm)] diagnosed with magnetic resonance (MR)-invisible disease or MR-visible disease using the external phased-array receiver. PATIENTS AND METHODS: Between 2007 and 2014, 110 patients with a FIGO clinical stage IA2-IB1 (<2 cm) cervical cancer underwent primary surgical treatment after external array coil T2W and DW MR imaging following the diagnostic biopsy procedure. RESULTS: The median histological size of MR-invisible vs. MR-visible diagnosis was 3±6.4 mm and 16±5.2 mm. Eighty-five of the 110 patients had histologically residual tumor. The sensitivity, specificity, PPV, and NPV of tumor diagnosis were 63.5%, 92.0%, 96.4%, and 42.6%, respectively. Histological estimates of 54 (49.1%) MR-invisible vs. 56 (50.9%) MR-visible diagnoses were identified as 23 true-negative (TN) and 31 false-negative (FN) vs. 54 true-positive (TP) and 2 false-positive (FP). The recurrence-free rate was 98.1% in the MR-invisible group and 91.1% in the MR-visible group. The overall survival rates were 100% and 92.9%, respectively. CONCLUSION: A preoperative MR-invisible diagnosis in early-stage cervical cancer patients led to a high probability of FN and was associated with underdiagnosis.

Successful term delivery cases of trans-abdominal cervicoisthmic cerclage performed at more than 18 weeks of gestation.

A 38-year-old nulliparous woman was referred to our clinic because of cervical incompetence at 19 weeks of gestation. Trans-abdominal cervicoisthmic cerclage was performed after failure of modified Shirodkar cerclage operation in the patient at 21 weeks of gestation via a laparotomic approach. Another 38-year-old patient, who underwent loop electrosurgical excision procedure conization for treatment of cervical dysplasia 4 years ago, presented for cervical incompetence. At 18 weeks of gestation, we performed trans-abdominal laparotomic cervicoisthmic cerclage without any post-operative complications. During antenatal follow-up, there were no obstetrical co-morbidities and finally she gave birth to a healthy infant at full term by cesarean section. We report two cases of women who underwent trans-abdominal cervicoisthmic cerclage surgery because of cervical incompetence as they were not suitable for transvaginal cervical cerclage. Both patients successfully maintained their pregnancy until full term after undergoing transabdominal cervicoisthmic cerclage at more than 18 weeks of gestation.