AJICAP-M: Traceless Affinity Peptide Mediated Conjugation Technology for Site-Selective Antibody-Drug Conjugate Synthesis.

A traceless site-selective conjugation method, "AJICAP-M", was developed for native antibodies at sites using Fc-affinity peptides, focusing on Lys248 or Lys288. It produces antibody-drug conjugates (ADCs) with consistent drug-to-antibody ratios, enhanced stability, and simplified manufacturing. Comparative in vivo assessment demonstrated AJICAP-M's superior stability over traditional ADCs. This technology has been successfully applied to continuous-flow manufacturing, marking the first achievement in site-selective ADC production. This manuscript outlines AJICAP-M's methodology and its effectiveness in ADC production.

Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification.

The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

Novel formats of antibody conjugates: recent advances in payload diversity, conjugation, and linker chemistry.

INTRODUCTION: In the field of bioconjugates, the focus on antibody - drug conjugates (ADCs) with novel payloads beyond the traditional categories of potent cytotoxic agents is increasing. These innovative ADCs exhibit various molecular formats, ranging from small-molecule payloads, such as immune agonists and proteolytic agents, to macromolecular payloads, such as oligonucleotides and proteins. AREAS COVERED: This review offers an in-depth exploration of unconventional strategies for designing conjugates with novel mechanisms of action and notable examples of approaches that show promising prospects. Representative examples of novel format payloads and their classification, attributes, and appropriate conjugation techniques are discussed in detail. EXPERT OPINION: The existing basic technologies used to manufacture ADCs can be directly applied to synthesize novel formatted conjugates. However, a wide variety of new payloads require the creation of customized technologies adapted to the unique characteristics of these payloads. Consequently, fundamental technologies, such as conjugation methods aimed at achieving high drug - antibody ratios and developing stable crosslinkers, are likely to become increasingly important research areas in the future.

AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugate Production.

The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the antibody-drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G (IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance the therapeutic index of the resultant ADCs. This methodology, termed "AJICAP", successfully modified Lys248 of native antibodies to produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla. However, the long reaction sequences, including the reduction-oxidation (redox) treatment, increased the aggregation level. In this manuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named "AJICAP second generation" without redox treatment utilizing a "one-pot" antibody modification reaction. The stability of Fc affinity reagents was improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248 conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs from several combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared. Furthermore, nontraditional ADC production, such as antibody-protein conjugates and antibody-oligonucleotide conjugates, were achieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates without antibody engineering.

MC180295 Inhibited Epstein-Barr Virus-Associated Gastric Carcinoma Cell Growth by Suppressing DNA Repair and the Cell Cycle.

DNA methylation of both viral and host DNA is one of the major mechanisms involved in the development of Epstein-Barr virus-associated gastric carcinoma (EBVaGC); thus, epigenetic treatment using demethylating agents would seem to be promising. We have verified the effect of MC180295, which was discovered by screening for demethylating agents. MC180295 inhibited cell growth of the EBVaGC cell lines YCCEL1 and SNU719 in a dose-dependent manner. In a cell cycle analysis, growth arrest and apoptosis were observed in both YCCEL1 and SNU719 cells treated with MC180295. MKN28 cells infected with EBV were sensitive to MC180295 and showed more significant inhibition of cell growth compared to controls without EBV infection. Serial analysis of gene expression analysis showed the expression of genes belonging to the role of BRCA1 in DNA damage response and cell cycle control chromosomal replication to be significantly reduced after MC180295 treatment. We confirmed with quantitative PCR that the expression levels of BRCA2, FANCM, RAD51, TOP2A, and CDC45 were significantly decreased by MC180295. LMP1 and BZLF1 are EBV genes with expression that is epigenetically regulated, and MC180295 could up-regulate their expression. In conclusion, MC180295 inhibited the growth of EBVaGC cells by suppressing DNA repair and the cell cycle.

Biological Evaluation of Maytansinoid-Based Site-Specific Antibody-Drug Conjugate Produced by Fully Chemical Conjugation Approach: AJICAP®.

BACKGROUND: Trastuzumab-emtansine (T-DM1, commercial name: Kadcyla) is well-known antibody-drug conjugate (ADC) and was first approved for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This molecular format consisting of trastuzumab and maytansinoid payload (emtansine) is very simple, however, T-DM1 has wide heterogeneity due to non-specific conjugation, lowering its therapeutic index (TI). METHODS: To overcome this issue during the chemical modification of the random conjugation approach to generate T-DM1, we developed a novel chemical conjugation technology termed "AJICAP®" for modification of antibodies in site-specific manner by IgG Fc-affinity peptide based reagents. RESULTS: In this study, we compared site-specific maytansinoid-based ADCs synthesized by AJICAP and T-DM1 in rat safety studies. The results indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index compared with that of commercially available T-DM1. Gram scale preparation of this AJICAP-ADC and the initial stability study are also described. CONCLUSIONS: Trastuzumab-AJICAP-maytansinoid produced by this unique chemical conjugation methodology showed higher stability and tolerability than commercially available T-DM1.

The bactericidal effect of far-UVC on ESBL-producing Escherichia coli.

Because extended-spectrum beta-lactamase (ESBL) infections can cause life-threatening disease and effective treatments need to be developed, we examined the bactericidal effect of far-ultraviolet C (far-UVC) light therapy on ESBL-producing Escherichia coli (E. coli). The bactericidal effect on 2 types of ESBL-producing E. coli was the same as that on the wild strain although the results of drug resistance tests varied among these strains. We believe that irradiation with far-UVC is effective in preventing infection by ESBL-producing E. coli in health care settings.

Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates.

To overcome a lack of selectivity during the chemical modification of native non-engineered antibodies, we have developed a technology platform termed "AJICAP" for the site-specific chemical conjugation of antibodies through the use of a class of IgG Fc-affinity reagents. To date, a limited number of antibody-drug conjugates (ADCs) have been synthesized via this approach, and no toxicological study was reported. Herein, we describe the compatibility and robustness of AJICAP technology, which enabled the synthesis of a wide variety of ADCs. A stability assessment of a thiol-modified antibody synthesized by AJICAP technology indicated no appreciable increase in aggregation or decomposition upon prolonged storage, indicating that the unexpectedly stable thiol intermediate has a great potential intermediate for payload or linker screening or large-scale manufacturing. Payload conjugation with this stable thiol intermediate generated several AJICAP-ADCs. In vivo xenograft studies indicated that the AJICAP-ADCs displayed significant tumor inhibition comparable to benchmark ADC Kadcyla. Furthermore, a rat pharmacokinetic analysis and toxicology study indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index, compared with stochastic conjugation technology. This is the first report of the therapeutic index estimation of site-specific ADCs produced by utilizing Fc affinity reagent conjugation. The described site-specific conjugation technology is a powerful platform to enable next-generation ADCs through reduced heterogeneity and enhanced therapeutic index.

In-situ Reverse Phased HPLC Analysis of Intact Antibody-Drug Conjugates.

The field of oncology has recently seen an exponential growth in antibody-drug conjugates (ADCs) as a biopharmaceutical class with seven ADCs being launched onto the market in the last ten years. Despite the increase in the industrial research and development of these compounds, their structural complexity and heterogeneity continue to present various challenges regarding their analysis including reaction monitoring. Robust and simple reaction monitoring analysis are in demand in the view of at-line in-process monitoring, and can instill control, confidence and reliability in the ADC manufacturing process. Aiming at providing chromatographic methods for conjugation monitoring, we evaluated herein the potential of utilizing reverse phase HPLC analysis, without sample pretreatment, for characterization of traditional cysteine-based ADCs. This analysis can be used for estimation of drug antibody ratio (DAR), which has shown the same trends and results as other well-established HPLC techniques. This methodology was also applied to three ADCs derived from three different antibodies. Additionally, we analyzed unpurified ADC samples existing in a complex reaction matrix and separated ADC species and payload compounds. This investigation was conducted using three different ADCs based on different payloads. The results described herein indicate the potential application of this RP-HPLC methodology in reaction monitoring studies.

Physical characteristics comparison between maytansinoid-based and auristatin-based antibody-drug conjugates.

Aim: Direct analytical comparison of two major drug-linkers in the antibody-drug conjugate (ADC) field was conducted. Methods: Four different analytical methods [AlogP calculation, reverse phase (RP) high-performance liquid chromatography (HPLC; RP-HPLC), size exclusion chromatography HPLC (SEC-HPLC), and differential scanning calorimetry (DSC)] were tested for this comparison. Results: Maytansinoid-based ADCs showed less hydrophobicity than auristatin-based ADCs. Regardless of the drug-linker and drug-to-antibody ratios (DARs), the stability detected by DSC was decreased by conjugation. Conclusions: The cost and time-efficient analytical comparison described in this manuscript may be useful information for an initial characterization of ADCs prior to detailed biological studies.

Intra-abdominal desmoid tumor mimicking local recurrence of renal cell carcinoma after laparoscopic partial nephrectomy.

A 64-year-old man had an intra-abdominal mass that was detected in a follow-up examination after laparoscopic partial nephrectomy for renal cell carcinoma (RCC). CT showed an enhanced mass of 2.5-cm diameter near the right kidney, where partial nephrectomy had been performed. Local recurrence of RCC with duodenum invasion was suspected, and excision was performed. The final pathological diagnosis of desmoid tumor differed from the preoperative diagnosis. Therefore, we report this case as a rare example of intra-abdominal desmoid tumor mimicking local recurrence of RCC. To our knowledge, this is the first report of intra-abdominal desmoid tumor after laparoscopic partial nephrectomy.

Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components.

The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.

Male squirting: Analysis of one case using color Doppler ultrasonography.

INTRODUCTION: There is no scientific evidence for the mechanism of male squirting, although the term is common in mass media. Here, we describe the first recording of male squirting using color Doppler ultrasonography. CASE PRESENTATION: We recruited a 25-year-old male volunteer who was able to have male squirting. A transrectal ultrasound probe was inserted into the rectum and male squirting was observed following normal ejaculation. With penis stimulation for a further 20 s after ejaculation, translucent misty fluids with a creatine level similar to that of urine came from the extraurethral orifice for about 60 s. Color Doppler ultrasonography recorded strong contraction of the prostate and pelvic striated muscles just before male squirting, and then the stream went from the urinary bladder through the prostatic urethra. CONCLUSION: In male squirting, urine in the bladder gushes out from the external urethral orifice due to strong contraction of the prostate and pelvic striated muscles.

Tadalafil for male lower urinary tract symptoms improves endothelial function.

OBJECTIVES: To investigate the effects of tadalafil on vascular endothelial function and cardiovascular risk in patients with prostatic hyperplasia. METHODS: Tadalafil 5 mg was given to 20 patients with prostatic hyperplasia for whom an α1-blocker was ineffective. Voiding symptoms and vascular endothelial function were investigated before and after 4 and 12 weeks of administration, using commercial tests for vascular function and vascular endothelial function. RESULTS: The participants had a median age of 65 years, a mean body mass index of 24.2 and a mean prostate volume of 36.2 mL measured using transabdominal sonography. Voiding symptoms were significantly improved by tadalafil, based on the International Prostate Symptom Score, quality of life index and overactive bladder symptom score (P < 0.05). There were also significant improvements in vascular function (change of brachial-ankle pulse wave velocity from 1701 [before] to 1657 [4 weeks tadalafil] and 1525 [10 weeks tadalafil] cm/s [P < 0.05]) and vascular endothelial function (change of reactive hyperemia index from 1.36 to 1.56 and 1.89 [P < 0.05]). The change in reactive hyperemia index was significantly correlated with International Prostate Symptom Score, quality of life index and brachial-ankle pulse wave velocity. CONCLUSIONS: The improvement in intrapelvic blood flow by tadalafil can result in improved vascular endothelial function, in addition to improvement of voiding symptoms. The change in reactive hyperemia index seems to correlate with the severity of voiding symptoms, with tadalafil being most effective in patients with mild voiding symptoms.

Practical application of color Doppler ultrasonography in patients with ejaculatory dysfunction.

We describe two cases in which dynamic analysis of ejaculation using color Doppler ultrasonography was useful in diagnosis of ejaculatory dysfunction and planning of therapy. The first patient was a 32-year-old man with a diagnosis of retrograde ejaculation. A bladder neck collagen injection was carried out, as the main cause was thought to be the bladder neck remaining open during ejaculation. The patient had antegrade ejaculation 1 week later. The second patient was a 48-year-old man with a diagnosis of anorgasmia accompanied by decreased seminal emission and insufficient function of the rhythmic pelvic striated muscles. The patient was prescribed etilefrine hydrochloride 15 mg/day. The symptom improved 2 weeks after starting this drug. These cases suggest that the use of color Doppler ultrasonography during ejaculation can improve the understanding of ejaculatory dysfunction and therapy for this condition.

Salvage high-dose-rate brachytherapy for local prostate cancer recurrence after radiotherapy - preliminary results.

OBJECTIVE: To assess the preliminary clinical results of salvage high-dose-rate brachytherapy (HDR-BT) applied in cases of suspected local recurrence or of residual tumour after radiotherapy. PATIENTS AND METHODS: The subjects were 11 patients who met the above conditions and underwent salvage HDR-BT between December 2006 and January 2009. The T stage at the initial treatment was T1c in three patients, T2 in three patients and T3 in five patients. Ten patients received HDR-BT ± electron beam radiation therapy and one patient received proton beam irradiation. Follow-up after the completion of salvage HDR-BT lasted 18-41 months (mean 29 months). A dose of 11.0 Gy radiation was delivered twice (22.0Gy in total), separated by a 6-h interval, on the day the applicators were inserted. RESULTS: Seven of the 11 cases remained in a biochemical non-evidence of disease state. The prostate-specific antigen (PSA) level continuously rose after salvage HDR-BT in three of the four other cases. Hormone administration was initiated in the four cases of PSA recurrence. No G3 or more severe events occurred, and the incidence of G2 was low during this study period. CONCLUSION: Of the 11 cases treated with salvage HDR-BT, PSA levels remained low in seven cases and the incidence of complications was also low. This suggests that salvage HDR-BT is effective as an option for treatment of local prostate cancer recurrence after radiotherapy.

Comparison of Two Different α1-Adrenoceptor Antagonists, Tamsulosin and Silodosin, in the Treatment of Male Lower Urinary Tract Symptoms Suggestive of Benign Prostatic Hyperplasia: A Prospective Randomized Crossover Study.

OBJECTIVES: We assessed the efficacy and safety of two α1-adrenoceptor antagonists, tamsulosin and silodosin, in the treatment of male lower urinary tract symptoms. METHODS: Men aged 50 years or older who had a total International Prostate Symptom Score (IPSS) of 8 or higher were enrolled in this study. Forty-six patients were randomized into two groups. Twenty-three patients were initially prescribed tamsulosin 0.2 mg once daily for 3 months, followed by silodosin 4 mg twice daily for 3 months (group T); the other group of 23 patients were initially prescribed silodosin, followed by tamsulosin (group S). Patients then switched to the alternative treatment after a 1-month clearance period. Evaluations included clinical determination of IPSS, quality-of-life index, maximum flow rate and postvoid residual urine volume before and after treatment. RESULTS: A total of 46 men, 23 in group T and 23 in group S, were treated and 41 (89.1%) completed the treatment. IPSS, quality-of-life index, maximum flow rate and postvoid residual urine volume were significantly improved in both groups after treatment. The changes in the total IPSS from baseline in groups S and T at 3 months were -6.6 and -7.5, respectively. There were no significant differences between the two groups. After taking both medications, 18 patients preferred silodosin, 11 preferred tamsulosin and others felt they had the same effects. Six and none patients experienced adverse events during silodosin and tamsulosin treatment, respectively. CONCLUSION: Two types of α1-adrenoceptor antagonists in the same individuals provide similar efficacy. Profiles and difference of each drug should be considered in making treatment choice.

Retroperitoneoscopic nephrectomy for treatment of a case of left single ectopic ureter accompanied by dysplastic kidney.

We report the case of a 7-year-old girl with a single ectopic ureter who was treated with retroperitoneoscopic nephrectomy for a chief complaint of urinary incontinence. Preoperative CT showed a contrasted dysplastic kidney of 1cm in the renal fossa and a left ureteral opening into the vagina. Retroperitoneoscopic left nephrectomy was conducted with opening of the lateroconal fascia to enable identification of the dysplastic kidney. No intraoperative complications were encountered. Urinary incontinence improved immediately after surgery. This case shows that a retroperitoneal approach can be used in nephrectomy if the position of the kidney can be determined preoperatively.

Effects of three types of alpha-1 adrenoceptor blocker on lower urinary tract symptoms and sexual function in males with benign prostatic hyperplasia.

OBJECTIVES: The aim of the present study was to explore the effects of three different types of alpha-1 adrenoceptor blockers (α1-blocker) on lower urinary tract symptoms (LUTS), erectile dysfunction (ED) and ejaculatory dysfunction (EjD) in patients with benign prostatic hyperplasia. METHODS: A total of 136 male LUTS patients aged 50-80 years with International Prostate Symptom Score (IPSS) ≥8 were enrolled. They were divided into three groups. Group S received silodosin at 4 mg twice a day; group T received tamsulosin at 0.2 mg once a day; and group N received naftopidil at 50 mg once a day. Assessment included IPSS, quality of life indexes (QOL), International Index of Erectile Function (IIEF-5), an ejaculation questionnaire, Qmax and post-void residual urine volume (PVR). These parameters were recorded at baseline, and at 1 and 3 months after treatment had ended. RESULTS: Mean IPSS and Qmax significantly improved after treatment in all groups without any significant difference among them. As for the IIEF-5 score, only group N significantly improved at 1 and 3 months. After treatment, 2.6 and 2.4% of patients complained of a de novo reduced volume of ejaculation in both groups T and N, respectively. Ten out of 41 patients (24.4%) complained of a total absence of antegrade ejaculation in group S after treatment. CONCLUSIONS: All three types of α1-blockers provided an objective and subjective improvement of LUTS in the present study population. However, erectile function only improved in patients treated with naftopidil and a higher rate of EjD was observed in those receiving silodosin. Because of their variable effects, we should consider the sexual dimension when prescribing α1-blockers for LUTS.