Prevalence and prognostic value of PD-L1 expression and tumor mutational burden in persistent, recurrent, or metastatic cervical cancer.

OBJECTIVE: To evaluate the prevalence and prognostic role of programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB) in patients with non-immunotherapy-treated advanced cervical cancer. METHODS: Clinical data were retrospectively collected from medical records between January 1, 2008, and December 31, 2016, at Asan Medical Center (Korea); archived tumor samples were assessed for PD-L1 expression (combined positive score [CPS] ≥1) and TMB (≥175 mutations/exome). Overall survival (OS) was defined as time from advanced diagnosis or initiation of first-line or second-line systemic therapy until death/last follow-up. The association of OS with PD-L1 expression and TMB were analyzed using the log-rank test and Cox proportional hazards model adjusted for covariates. RESULTS: Of 267 patients, 76.0% had squamous cell carcinoma (SCC), 24.0% had adenocarcinoma (AC)/adenosquamous carcinoma (ASC), 64.4% had PD-L1 CPS ≥1, and 32.6% had TMB ≥175 mutations/exome. PD-L1 CPS ≥1 and TMB ≥175 mutations/exome were more prevalent in SCC than in AC/ASC (73.9% and 37.2% vs. 34.4% and 17.7%). There was no association between OS and PD-L1 expression (CPS ≥1 vs. <1: adjusted hazard ratio [HR]=1.14; 95% confidence interval [CI]=0.84-1.53 from advanced diagnosis); OS trended shorter for the subgroup with TMB ≥175 versus <175 mutations/exome (adjusted HR=1.29; 95% CI=0.95-1.75). CONCLUSION: Retrospective analysis of non-immunotherapy-treated patients with advanced cervical cancer demonstrated a higher prevalence of PD-L1 CPS ≥1 and TMB ≥175 mutations/exome in SCC versus AC/ASC. PD-L1 CPS ≥1 was not associated with OS; TMB ≥175 mutations/exome showed a trend toward shorter OS. Additional studies are needed to confirm these findings.

Expression and scale-up production of recombinant human papillomavirus type 52 L1 protein in methylotrophic yeast Hansenula polymorpha.

BACKGROUND: Human papillomavirus (HPV) vaccination is one of the crucial national vaccination programs aimed at reducing the prevalence of the diseases associated with HPV infections, which continue to pose a global health concern. However, a significant disparity exists in the distribution of HPV vaccine, particularly in low-middle income countries where the cost of HPV vaccine becomes a major obstacle. Thus, it is essential to ensure the availability of an economically feasible HPV vaccine, necessitating immediate efforts to enhance the cost-effectiveness of vaccine production. This study aimed to develop an efficient production system for the recombinant HPV type 52 L1 protein as HPV vaccine material using methylotrophic yeast Hansenula polymorpha expression system. RESULTS: This study presents an in-depth examination of the expression and scale-up production of HPV type 52 L1 protein using DASGIP® parallel bioreactor system. The pHIPX4 plasmid, which is regulated by the MOX promoter, generates stable clones that express the target protein. Cultivation employing the synthetic medium SYN6(10) with controlled parameters (e.g. temperature, pH, feeding strategy, and aeration) produces 0.15 µg/mL of HPV type 52 L1 protein, suggesting a possibility for scaling up to a higher production level. CONCLUSION: The scale-up production of HPV type 52 L1 protein using Hansenula polymorpha expression system described in this study provides an opportunity for an economical manufacturing platform for the development of the HPV vaccine.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies.

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200-350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251-305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262-279. Amino acid 262-279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262-273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

Human Papilloma Virus-Infected Cells.

Human papillomavirus (HPV) is associated with infection of different tissues, such as the cervix, anus, vagina, penis, vulva, oropharynx, throat, tonsils, back of the tongue, skin, the lungs, among other tissues. HPV infection may or may not be associated with the development of cancer, where HPVs not related to cancer are defined as low-risk HPVs and are associated with papillomatosis disease. In contrast, high-risk HPVs (HR-HPVs) are associated with developing cancers in areas that HR-HPV infects, such as the cervix. In general, infection of HPV target cells is regulated by specific molecules and receptors that induce various conformational changes of HPV capsid proteins, allowing activation of HPV endocytosis mechanisms and the arrival of the HPV genome to the human cell nucleus. After the transcription of the HPV genome, the HPV genome duplicates exponentially to lodge in a new HPV capsid, inducing the process of exocytosis of HPV virions and thus releasing a new HPV viral particle with a high potential of infection. This infection process allows the HPV viral life cycle to conclude and enables the growth of HPV virions. Understanding the entire infection process has been a topic that researchers have studied and developed for decades; however, there are many things to still understand about HPV infection. A thorough understanding of these HPV infection processes will allow new potential treatments for HPV-associated cancer and papillomatosis. This chapter focuses on HPV infection, the process that will enable HPV to complete its HPV life cycle, emphasizing the critical role of different molecules in allowing this infection and its completion during the HPV viral life cycle.

Protective Effects of Melatonin in High-Fat Diet-Induced Hepatic Steatosis via Decreased Intestinal Lipid Absorption and Hepatic Cholesterol Synthesis.

BACKGRUOUND: The preventative effect of melatonin on the development of obesity and the progression of fatty liver under a high-fat diet (HFD) has been well elucidated through previous studies. We investigated the mechanism behind this effect regarding cholesterol biosynthesis and regulation of cholesterol levels. METHODS: Mice were divided into three groups: normal chow diet (NCD); HFD; and HFD and melatonin administration group (HFD+M). We assessed the serum lipid profile, mRNA expression levels of proteins involved in cholesterol synthesis and reabsorption in the liver and nutrient transporters in the intestines, and cytokine levels. Additionally, an in vitro experiment using HepG2 cells was performed. RESULTS: Expression of hepatic sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and low-density lipoprotein receptor (LDLR) demonstrated that melatonin administration significantly reduces hepatic cholesterol synthesis in mice fed an HFD. Expression of intestinal sodium-glucose transporter 1 (SGLT1), glucose transporter 2 (GLUT2), GLUT5, and Niemann-pick C1-like 1 (NPC1L1) demonstrated that melatonin administration significantly reduces intestinal carbohydrate and lipid absorption in mice fed an HFD. There were no differences in local and circulatory inflammatory cytokine levels among the NCD, HFD, and HFD+M group. HepG2 cells stimulated with palmitate showed reduced levels of SREBP, LDLR, and HMGCR indicating these results are due to the direct mechanistic effect of melatonin on hepatocytes. CONCLUSION: Collectively, these data indicate the mechanism behind the protective effects of melatonin from weight gain and liver steatosis under HFD is through a reduction in intestinal caloric absorption and hepatic cholesterol synthesis highlighting its potential in the treatment of obesity and fatty liver disease.

Human papilloma virus presence and its physical status in primary pterygium.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, overgrowth of fibrovascular tissue, often with a wing-like appearance, from the conjunctiva over the cornea. It is composed of an epithelium and highly vascular, sub-epithelial, loose connective tissue. There is much debate surround the pathogenesis of pterygium and a number of theories have been put forward including genetic instability, cellular proliferation, inflammatory influence, and degeneration of connective tissue, angiogenesis, aberrant apoptosis and viral infection. At present, the involvement of human papillomavirus (HPV) in the genesis of pterygium is controversial, as have reported that HPV is present in 58% of cases, while others have failed to detect HPV in pterygium. In this study, we evaluated the presence and viral genotype of HPV DNA in pterygia and healthy conjunctiva sample, and virus integration into the cellular genome. Forty primary pterygia samples and 12 healthy conjunctiva samples were analyzed to HPV DNA presence by polymerase chain reaction, using MY09/MY11 primers of HPV-L1 gene. Viral genotype was identified by DNA sequence analysis of this amplicon. HPV integration into the cellular genome was analyzed by western blot detecting HPV-L1 capsid protein. Presence of HPV was observed in 19 of the 40 pterygia samples. In contrast, healthy conjunctiva samples were negative. To determine virus type, sequence analyses were performed. Interestingly, 11 out of the 19-pterygium samples were identified as HPV-11 type, meanwhile, the remaining 8 pterygium samples were identified as HPV-18. HPV-L1 capsid protein were found only in 3 out of the 10 samples studied. In conclusion, our study identified the presence of HPV DNA exclusively in pterygium samples and described HPV-11 and -18 genotypes. Our results suggest that HPV may be involved in the pathogenesis of pterygium. On the other hand, the expression of the L1-HPV protein suggests viral integration into the cellular genome.

Predictive Capability of PD-L1 Protein Expression for Patients With Advanced NSCLC: Any Differences Based on Histology?

Lung cancer is responsible for 1.8 million annual deaths. Non-small cell lung cancers (NSCLC) represent 85% of lung cancer tumors. While surgery is an effective early-stage treatment, the majority of newly identified US lung cancer cases are stage III/IV. Immunotherapy, using programmed death-ligand 1 (PD-L1) or programmed death 1 (PD-1) receptor antibody therapeutics, has increased survival for patients with NSCLC. PD-L1 protein expression is widely used as a predictive biomarker informing treatment decisions. However, only a minority of patients (27%-39%) respond to PD-L1/PD-1 treatment. PD-L1 protein expression by immunohistochemistry assay has deficiencies in identifying responding and refractory patients. Given the different characteristics of squamous and nonsquamous NSCLC, the predictability of PD-L1 levels in determining which patients would benefit from immunotherapy could vary between the 2 histologies. We analyzed 17 phase-III clinical studies and a retrospective study to determine if the predictive capability of PD-L1 expression varies between squamous and nonsquamous NSCLC. For patients with NSCLC treated with mono or dual-immune checkpoint inhibitors (ICI), PD-L1 expression was more predictive of benefit for patients with nonsquamous NSCLC than squamous NSCLC. Patients with nonsquamous histology and PD-L1 high tumor proportion scores (TPS) survived 2.0x longer compared to those with low TPS, when treated with monotherapy ICI. Among patients with squamous NSCLC, that difference was 1.2 to 1.3x. For patients treated with ICIs and chemotherapy, there was no clear difference in the predictive value of PD-L1 levels between histologies. We encourage future researchers to analyze the predictability of PD-L1 biomarker expression separately for squamous and nonsquamous NSCLC.

Results from the IMpower132 China cohort: Atezolizumab plus platinum-based chemotherapy in advanced non-small cell lung cancer.

BACKGROUND: The global Phase III IMpower132 study evaluating atezolizumab plus pemetrexed and carboplatin or cisplatin (APP) versus pemetrexed plus carboplatin or cisplatin (PP) for first-line treatment of non-squamous advanced non-small cell lung cancer (NSCLC) met its co-primary progression-free survival (PFS) endpoint at the primary analysis in the intention-to-treat (ITT) population. Although the co-primary overall survival (OS) endpoint was not met, numerical OS improvement favoring APP over PP was observed at the final analysis. We report primary results for Chinese patients in IMpower132. METHODS: Treatment-naive Chinese patients with non-squamous stage IV EGFR/ALK mutation-negative NSCLC were randomized 1:1 to receive 4 or 6 cycles of APP or PP, followed by maintenance atezolizumab plus pemetrexed or pemetrexed. Co-primary endpoints were investigator-assessed PFS and OS. RESULTS: The ITT population included 163 Chinese patients (82 in the APP arm and 81 in the PP arm). At data cutoff (median follow-up, 11.7 months), the median PFS in the APP and PP arms was 8.3 and 5.8 months, respectively; the unstratified hazard ratio (HR) was 0.73 (95% CI: 0.50, 1.08). At the interim OS analysis, median OS was not estimable in either arm; the unstratified HR was 0.70 (95% CI: 0.40, 1.24). No new safety signals were observed. CONCLUSION: Among Chinese patients in IMpower132, PFS benefit was seen with APP versus PP. Though interim OS data were immature, there was a trend toward OS benefit favoring APP versus PP. The safety profile of the APP was consistent with the known risks of the individual treatment components. CLINICALTRIALS: gov: NCT02657434.

Adjuvant atezolizumab in Japanese patients with resected stage IB-IIIA non-small cell lung cancer (IMpower010).

The global phase 3 IMpower010 study evaluated adjuvant atezolizumab versus best supportive care (BSC) following platinum-based chemotherapy in patients with resected stage IB-IIIA non-small cell lung cancer (NSCLC). Here, we report a subgroup analysis in patients enrolled in Japan. Eligible patients had complete resection of histologically or cytologically confirmed stage IB (tumors ≥4 cm)-IIIA NSCLC. Upon completing 1-4 cycles of adjuvant cisplatin-based chemotherapy, patients were randomized 1:1 to receive atezolizumab (fixed dose of 1200 mg every 21 days; 16 cycles or 1 year) or BSC. The primary endpoint of the global IMpower010 study was investigator-assessed disease-free survival, tested hierarchically first in patients with stage II-IIIA NSCLC whose tumors expressed programmed death-ligand 1 (PD-L1) on ≥1% of tumor cells, then in all randomized patients with stage II-IIIA NSCLC, and finally in the intention-to-treat (ITT) population (stage IB-IIIA NSCLC). Safety was evaluated in all patients who received atezolizumab or BSC. The study comprised 149 enrolled patients in three populations: ITT (n = 117; atezolizumab, n = 59; BSC, n = 58), all-randomized stage II-IIIA (n = 113; atezolizumab, n = 56; BSC, n = 57), and PD-L1 tumor cells ≥1% stage II-IIIA (n = 74; atezolizumab, n = 41; BSC, n = 33). At the data cutoff date (January 21, 2021), a trend toward disease-free survival improvement with atezolizumab vs BSC was observed in the PD-L1 tumor cells ≥1% stage II-IIIA (unstratified hazard ratio [HR], 0.52; 95% confidence interval [CI], 0.25-1.08), all-randomized stage II-IIIA (unstratified HR, 0.62; 95% CI, 0.35-1.11), and ITT (unstratified HR, 0.61; 95% CI, 0.34-1.10) populations. Atezolizumab-related grade 3/4 adverse events occurred in 16% of patients; no treatment-related grade 5 events occurred. Adjuvant atezolizumab showed disease-free survival improvement and a tolerable toxicity profile in Japanese patients in IMpower010, consistent with the global study results.

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3.

BACKGROUND: A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. RESULTS: The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs. CONCLUSION: Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.

Combined truncations at both N- and C-terminus of human papillomavirus type 58 L1 enhanced the yield of virus-like particles produced in a baculovirus system.

Human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs) produced in baculovirus system are highly immunogenic, but the relatively high production cost limits its application in the development of broad-spectrum vaccines. Here we report a novel method for enhancing VLP production in this system. We incorporated respectively 4, 8 or 13 residues truncation mutations in the N-terminus of L1ΔC, a C-terminal 25-residue-deleted L1 of HPV58, to construct three mutants. After expression in Sf9 cells, L1ΔN4C exhibited 2.3-fold higher protein production, 2.0-fold mRNA expression and lower rate of mRNA decay, compared to L1ΔC. More importantly, L1ΔN4C protein was easily purified by two-step chromatography with a VLP yield of up to 60 mg/L (purity > 99 %), 5-fold that of L1ΔC, whereas L1ΔN8C and L1ΔN13C behaved similarly to L1ΔC either in protein or mRNA expression. Moreover, L1ΔN4C VLPs showed similar binding activities with six HPV58 neutralizing monoclonal antibodies and induced comparable level of neutralizing antibody in mice to that of L1ΔC VLPs. Our results demonstrate that certain N- and C-terminal truncations of HPV58 L1 can enhance VLP yield. This method may be used to reduce production costs of other L1VLPs or chimeric VLPs to developing pan-HPV vaccines using baculovirus system.

Expression of Programmed Cell Death-L1 (PD-L1) Protein and Mismatch Repair Mutations in Orbital Tumours-a Pilot Study.

PURPOSE: Programmed cell death protein 1 (PD-1) and DNA mismatch repair (MMR) deficiency play an important role in tumour progression and response to treatment.Both markers have been studied in some ocular tumours but little is known about these markers in orbital tumours. This pilot study reports on PD-L1 expression and MMR mutations using next generation sequencing (NGS) in specific orbital tumours. METHODS: We reviewed surgical specimens from patients with rhabdomyosarcoma, adenoid cystic carcinoma (ACC), pleomorphic adenoma (PA) and biopsy negative tissue from orbital tumours used as a control. immunohistochemistry (IHC) was performed on Formalin fixed paraffin embedded tissue using a PD-L1 antibody. DNA was extracted for targeted gene panel NGS of the MMR genes PMS2, MLH1, MSH6 and MSH2. RESULTS: The study included 17 orbital specimens. Scattered membrane PD-L1 staining was noted in 3/6 rhabdomyosarcoma specimens without an accompanying lymphocytic infiltrate. PD-L1 immunostaining was absent in 3/3 ACC, and 5/6 PA specimens. PD-L1 immunostaining was not detected in 2/2 control specimens. 4/17 samples shared the same pathogenic mutation in the MLH1 gene, including 3/6 rhabdomyosarcoma and 1/3 ACC samples. 1/6 PA samples had a mutation in MSH6. CONCLUSIONS: Our study demonstrated scattered, non-quantifiable or absent PD-L1 staining in a limited sample of orbital tumours suggesting that PD-1/PD-L1 inhibitor therapy may not be useful in treatment of malignant orbital tumours (rhabdomyosarcoma and ACC) when refractory to conventional therapy. Our pilot study suggest that PD-L1/MMR axis might not play a major role in the pathogenesis of primary orbital tumour.

First-line pembrolizumab vs chemotherapy in metastatic non-small-cell lung cancer: KEYNOTE-024 Japan subset.

This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE-024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non-small-cell lung cancer without EGFR/ALK alterations and a programmed death-ligand 1 (PD-L1) tumor proportion score of 50% or greater evaluated clinical outcomes among patients enrolled in Japan. Treatment consisted of pembrolizumab 200 mg every 3 weeks (35 cycles) or platinum-based chemotherapy (four to six cycles). The primary end-point was progression-free survival; secondary end-points included overall survival and safety. Of 305 patients randomized in KEYNOTE-024 overall, 40 patients were enrolled in Japan (all received treatment: pembrolizumab, n = 21; chemotherapy, n = 19). The hazard ratio (HR) for progression-free survival by independent central review (data cut-off date, 10 July 2017) was 0.25 (95% confidence interval [CI], 0.10-0.64; one-sided, nominal P = .001). The HR for overall survival (data cut-off date, 15 February 2019) was 0.39 (95% CI, 0.17-0.91; one-sided, nominal P = .012). Treatment-related adverse events occurred in 21/21 (100%) pembrolizumab-treated and 18/19 (95%) chemotherapy-treated patients; eight patients (38%) and nine patients (47%), respectively, had grade 3-5 events. Immune-mediated adverse events and infusion reactions occurred in 11 patients (52%) and four patients (21%), respectively; four patients (19%) and one patient (5%), respectively, had grade 3-5 events. Consistent with results from KEYNOTE-024 overall, first-line pembrolizumab improved progression-free survival and overall survival vs chemotherapy with manageable safety among Japanese patients with metastatic non-small-cell lung cancer without EGFR/ALK alterations and a PD-L1 tumor proportion score of 50% or greater. The trial is registered with ClinicalTrials.gov: NCT02142738.

PD-L1 Protein Expression and Gene Amplification Correlate with the Clinicopathological Characteristics and Prognosis of Lung Squamous Cell Carcinoma.

PURPOSE: To investigate PD-L1 protein expression and gene amplification in lung squamous cell carcinoma (LUSC) and analyse their correlation with the clinicopathological characteristics and prognosis of LUSC patients. PATIENTS AND METHODS: Tissue samples from 164 LUSC patients were collected. PD-L1 protein was detected by immunochemistry (IHC), and PD-L1 gene amplification was investigated by fluorescence in situ hybridization in LUSC patients. RESULTS: The positive expression rate of PD-L1 in LUSC was 47.6% (78/164), and the amplification rate of PD-L1 was 6.7% (11/164); both rates were higher than those of paratumor tissue. Both PD-L1 positive expression and gene amplification were correlated with clinical stage and lymph node metastasis (P<0.05). PD-L1 protein expression, PD-L1 gene amplification, late stage, lymph node metastasis and distant metastasis were significantly correlated with the prognosis of patients. Among these factors, late stage, lymph node metastasis, PD-L1 protein expression and PD-L1 gene amplification were independent prognostic factors for LUSC. CONCLUSION: Positive PD-L1 protein expression and gene amplification are involved in the malignant progression and metastasis of LUSC. Both PD-L1 protein expression and gene amplification are associated with poor prognosis.

Differential Antibody Response against Conformational and Linear Epitopes of the L1 Proteins from Human Papillomavirus Types 16/18 Is Observed in Vaccinated Women or with Uterine Cervical Lesions.

Antibodies against the Human Papillomavirus (HPV) L1 protein are associated with past infections and related to the evolution of the disease, whereas antibodies against L1 Virus-Like Particles (VLPs) are used to follow the neutralizing antibody response in vaccinated women. In this study, serum antibodies against conformational (VLPs) and linear epitopes of HPV16/18 L1 protein were assessed to distinguish HPV-vaccinated women from those naturally infected or those with uterine cervical lesions. The VLPs-16/18 were generated in baculovirus, and L1 proteins were obtained from denatured VLPs. Serum antibodies against VLPs and L1 proteins were evaluated by ELISA. The ELISA-VLPs and ELISA-L1 16/18 assays were validated with a vaccinated women group by ROC analysis and the regression analysis to distinguish the different populations of female patients. The anti-VLPs-16/18 and anti-L1-16/18 antibodies effectively detect vaccinated women (AUC = 1.0/0.79, and 0.94/0.84, respectively). The regression analysis showed that anti-VLPs-16/18 and anti-L1-16/18 antibodies were associated with the vaccinated group (OR = 2.11 × 108/16.50 and 536.0/49.2, respectively). However, only the anti-L1-16 antibodies were associated with the high-grade lesions and cervical cancer (CIN3/CC) group (OR = 12.18). In conclusion, our results suggest that anti-VLPs-16/18 antibodies are effective and type-specific to detect HPV-vaccinated women, but anti-L1-16 antibodies better differentiate the CIN3/CC group. However, a larger population study is needed to validate these results.

Pembrolizumab plus pemetrexed-platinum for metastatic nonsquamous non-small-cell lung cancer: KEYNOTE-189 Japan Study.

Pembrolizumab plus pemetrexed-platinum significantly improved overall survival (OS) and progression-free survival (PFS) with manageable safety compared with placebo plus pemetrexed-platinum in patients with previously untreated metastatic nonsquamous non-small-cell lung cancer (NSCLC) without EGFR/ALK alterations in the global, randomized, double-blind, phase 3 KEYNOTE-189 study. We present results of Japanese patients enrolled in the KEYNOTE-189 global and Japan extension studies. Patients were randomized 2:1 to intravenous pembrolizumab 200 mg or placebo every 3 weeks (Q3W) for up to 35 cycles. All patients received pemetrexed 500 mg/m2 plus the investigator's choice of cisplatin or carboplatin Q3W for four cycles, followed by maintenance pemetrexed 500 mg/m2 Q3W (all intravenous). Co-primary endpoints were OS and PFS. Forty Japanese patients enrolled (pembrolizumab, n = 25; placebo, n = 15). At data cutoff (20 May 2019; median time from randomization to data cutoff, 18.5 [range, 14.7-38.2] months), the median OS was not reached in the pembrolizumab plus pemetrexed-platinum arm; the median OS was 25.9 (95% confidence interval [CI], 11.9-29.0) months in the placebo plus pemetrexed-platinum arm (hazard ratio [HR] .29; 95% CI, .07-1.15). The median (95% CI) PFS was 16.5 (8.8-21.1) compared with 7.1 (4.7-21.4) months (HR, .62; 95% CI, .27-1.42), respectively. There were no grade 5 adverse events (AE). Grade 3/4 AE occurred in 72% vs 60% of patients in the pembrolizumab vs placebo arms; 40% vs 20% had immune-mediated AE, and 4% vs 0% had infusion reactions. Efficacy and safety outcomes were similar to those from the global study and support first-line therapy with pembrolizumab plus pemetrexed-platinum in Japanese patients with nonsquamous NSCLC without EGFR/ALK alterations.

Development of a High-Throughput, Compound-Multiplexed Fluorescence Polarization Assay to Identify ATG5-ATG16L1 Protein-Protein Interaction Inhibitors.

Macroautophagy is a catabolic process wherein cytosolic cargo is engulfed in an autophagosome that fuses with a lysosome to degrade the cargo for recycling. Autophagy maintains cellular homeostasis and is involved in a myriad of illnesses ranging from cancer to neurodegenerative diseases, but its therapeutic potential remains elusive due to a lack of potent and specific autophagy modulators. To identify specific inhibitors of early autophagy, a target-based, compound-multiplexed, fluorescence polarization, high-throughput screen that targets the ATG5-ATG16L1 protein-protein interaction was developed. This interaction is critical for the formation of LC3-II, which is involved in phagophore maturation, and its disruption should inhibit autophagy. This assay is based on the polarization of light emitted by a fluorescent rhodamine tag conjugated to a peptide corresponding to the N-terminal region of ATG16L1 (ATG16L1-N). It was confirmed that this peptide binds specifically to ATG5, and the assay was validated by rapidly screening 4800 molecules through compound multiplexing. Through these initial screening efforts, a molecule was identified that disrupts the ATG5-ATG16L1 protein-protein interaction with micromolar potency, and this molecule will serve as a starting point for chemical optimization as an autophagy inhibitor.

Identification of the mimotopes within the major capsid protein L1 of human papillomavirus types 18 and 45, using neutralizing monoclonal antibodies.

Persistent infection with high-risk mucosal human papillomavirus (HPV) types has much association with the development of cervical cancer. The major capsid protein L1 has been confirmed to be a major candidate antigen for the development of vaccines. Here, the HPV18 L1 protein was successfully expressed and purified, then nine anti-HPV18 L1 monoclonal antibodies were prepared. Four neutralizing monoclonal antibodies (NmAbs) were identified by using hemagglutination inhibition assay and pseudovirus based neutralization assay. The results of Dot-ELISA, Western blot and indirect immunofluorescence assay showed that the neutralizing antibodies could cross-react with HPV16/18/45/31/33/58/35/39 L1. The mimotopes on HPV18/45 L1 proteins were identified and analyzed by using both phage display and Bioinformatics tool. The B cell epitopes 43-54 aa and 116-126 aa of HPV18 L1 protein, the B cell epitope 381-389 aa of HPV45 L1 protein, and the mimotopes epitope of HPV45 L1 protein were identified by peptide-ELISA and competitive ELISA. The results of PyMOL and Pepitope server analysis indicated that epitopes recognized by NmAbs 7F4, 5A6, 3G11, and 2F5 are located on the surface of L1 VLPs. The results of this study enriched the library of HPV neutralizing antibodies, revealed the mechanism of antibody neutralization, might open new perspectives on the antibody-antigen reaction and have important implications for the development of novel HPV vaccines.

Ultrasensitive electrochemical determination of the cancer biomarker protein sPD-L1 based on a BMS-8-modified gold electrode.

This work describes the modification of a gold electrode with the BMS-8 compound that interacts with the Programmed Death-Ligand 1 (PD-L1), an immune checkpoint protein. The results show that we can confirm the presence of the sPD-L1 in the concentration range of 10-18 to 10-8 M using electrochemical impedance spectroscopy (EIS) with a limit of detection (LOD) of 1.87 × 10-14 M for PD-L1 (S/N = 3.3) and at a concentration of 10-14 M via cyclic voltammetry (CV). Additionally, high-resolution X-ray photoelectron spectroscopy (XPS), contact angle, and surface free energy measurements were applied to confirm the functionalization of the electrode. We investigated the selectivity of the electrode for other proteins: Programmed Death-1 (PD-1), cluster of differentiation 160 (CD160), and B- and T-lymphocyte attenuator (BTLA) at concentrations of 10-8 M. Differentiation between PD-L1 and PD-1 was achieved based on the analysis of the capacitance effect frequency dispersion at the surface of the modified Au electrode with BMS-8 after incubation at various concentrations of PD-L1 and PD-1 proteins in the range of 10-18 to 10-8 M. Significant differences were observed in the heterogeneity of PD-L1 and PD-1. The results of the quasi-capacitance studies demonstrate that BMS-8 strongly and specifically interacts with the PD-L1 protein.

Simultaneous determination of capsid proteins in nine-valent human papilloma virus vaccines by liquid chromatography tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method was developed to determine nine types of capsid proteins simultaneously in nine-valent human papillomavirus vaccines. Signature peptides were optimized in terms of specificity, repeatability, determination accuracy and sensitivity. As a result, three signature peptides per capsid protein were obtained. The linear calibration curves were achieved in the range of 11.6-373.6 nmol/L (R2  > 0.998). Compared to our previous liquid chromatography-tandem mass spectrometry method, the current method was more sensitive (3.18-fold) and it can be used for quality evaluation of nine-valent human papillomavirus vaccines, unlike the previous method, which could only be used for bivalent human papillomavirus vaccines. Then, they were utilized to determine nine types of capsid proteins in nine-valent human papillomavirus vaccines from four different manufactures. Intraday and interday precision values for the determination of capsid proteins in nine-valent human papillomavirus vaccines were less than 6.8 and 9.1%, respectively. Recovery rates of all capsid proteins investigated were in the range of 80-120%. In addition, the current assay was used for determination of free capsid protein in nine-valent human papilloma virus vaccines, and the results were used to evaluate the adsorption rate of the adjuvant.