Clinical Development and Initial Approval of Novel Immune Checkpoint Inhibitors in Oncology: Insights From a Global Regulatory Perspective.

Immune checkpoint inhibitors (ICI) have demonstrated meaningful patterns of clinical efficacy across various cancers. During their development, novel regulatory strategies and clinical design approaches were explored. This metrics-based narrative review examines submission strategies and clinical evidence expectations of the US, European, and Japanese drug agencies, as well as their impact on approval and overall development times. Also discussed is the role of emerging clinical science and biomarker evaluation to get the first six ICI initially approved.

Multiple antigenic peptides as vaccine platform for the induction of humoral responses against dengue-2 virus.

Dengue is an important agent of human disease for which no licensed vaccine is available to the public. We used multiple antigenic peptides (MAPs) as an antigen carrier for the development of subunit vaccines against dengue-2 virus (DEN-2). Commercially available software (MacVector 7.0) was used to identify potential antigenic B-cell epitopes of E-glycoprotein. A total of 60 BALB/c mice were immunized with 12 recombinant DEN-2-specific MAPs and the humoral immune response was assessed by anti-DEN-2 ELISA and PRNT50 assays. Anti-DEN-2 ELISA showed high levels of anti-DEN-2 antibodies and post-immune sera reduced viral infectivity and prevented infection of monkey kidney cells (LLC-MK2) with live DEN-2 virus. Seven neutralizing DEN-2 epitopes were identified that generated PRNT50 titers of up to 1:160. Our findings show that the MAP platform can be used as an antigen-presenting platform for dengue vaccine development.

Parvovirus B19 empty capsids as antigen carriers for presentation of antigenic determinants of dengue 2 virus.

For the production of dengue-vaccine candidates, empty capsids, or virus-like particles (VLPs), of parvovirus B19 that carry dengue 2-specific epitopes were employed as antigen carriers. Two epitopes (comprising amino acids 352-368 and 386-397) of domain BIII of the envelope glycoprotein were chosen to produce recombinant B19 VLPs for immunization of BALB/c mice. Serum samples from immunized mice revealed that recombinant B19 VLPs elicited strong humoral immune responses. In summary, this B19 VLP-vaccine platform produced high (> or =2.0 x 10(5)) anti-dengue 2 titers and robust (< or =1 120) 50%-plaque-reduction neutralization test (PRNT(50)) titers, which effectively neutralized live dengue 2 virus in PRNT(50) assays.

Recombinant viral-like particles of parvovirus B19 as antigen carriers of anthrax protective antigen.

Viral-like particles (VLPs) of parvovirus B19 were employed as an antigen carrier to present antigenic determinants of Bacillus anthracis. The small-loop peptide and the full-length domain 4 of protective antigen (PA) were chosen as immunogens for presentation on the VLP-capsid surface and subsequent immunization of BALB/c mice. The recombinant VLPs induced anti-PA IgG titers of up to 2.5 x 10(4). Neutralization assays showed that the recombinant VLPs elicited neutralizing anti-PA antibody titers of up to 1:400 and showed potential for the prevention of lethal toxin-induced mortality of mouse-macrophage cells (RAW264.7). In postimmune sera, no anti-PA titers were detected against synthetic small-loop peptide. Recombinant VLPs demonstrated the capacity to retain the immunogenicity of the displayed microbial PA-epitopes and elicited robust levels of anti-PA antibody titers. These findings suggest that the recombinant VLPs of parvovirus B19 have potential as an additional tool in the development of sub-unit vaccines.

Changes in mumps virus gene sequence associated with variability in neurovirulent phenotype.

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.

Identification of a new genotype H wild-type mumps virus strain and its molecular relatedness to other virulent and attenuated strains.

A single clinical isolate of mumps virus designated 88-1961 was obtained from a patient hospitalized with a clinical history of upper respiratory tract infection, parotitis, severe headache, fever and lymphadenopathy. We have sequenced the full-length genome of 88-1961 and compared it against all available full-length sequences of mumps virus. Based upon its nucleotide sequence of the SH gene 88-1961 was identified as a genotype H mumps strain. The overall extent of nucleotide and amino acid differences between each individual gene and protein of 88-1961 and the full-length mumps samples showed that the missense to silent ratios were unevenly distributed. Upon evaluation of the consensus sequence of 88-1961, four positions were found to be clearly heterogeneous at the nucleotide level (NP 315C/T, NP 318C/T, F 271A/C, and HN 855C/T). Sequence analysis revealed that the amino acid sequences for the NP, M, and the L protein were the most conserved, whereas the SH protein exhibited the highest variability among the compared mumps genotypes A, B, and G. No identifying molecular patterns in the non-coding (intergenic) or coding regions of 88-1961 were found when we compared it against relatively virulent (Urabe AM9 B, Glouc1/UK96, 87-1004 and 87-1005) and non-virulent mumps strains (Jeryl Lynn and all Urabe Am9 A substrains).

Stability of the prion protein-encoding (PRNP) gene in HeLa cells.

To assess the risk of the de novo emergence of the agent of transmissible spongiform encephalopathies in cultured cells, we examined the stability of the prion protein-encoding (PRNP) gene in HeLa cells and in cultures contaminated with HeLa cells that have been passaged extensively for over 50 years. Various sub-lineages of HeLa cells showed that some contained a mixture of a truncated PRNP gene (R3-R4 deletion) and a full-length PRNP gene, while others were homozygous for the R3-R4 deletion. That finding suggests that the progenitor of several popular sub-lineages of HeLa must have lost part or all of chromosome 20 early in the history of HeLa cells. No mutations were found in the PRNP genes. We conclude that the spontaneous appearance of mutations leading to expression of abnormal prion proteins in continuously passaged heteroploid cell lines is unlikely to pose a substantial risk for the safe production of biologicals in such cells.

Sequence diversity of Jeryl Lynn strain of mumps virus: quantitative mutant analysis for vaccine quality control.

The Jeryl Lynn strain of mumps vaccine live (MVL) was developed in 1966 by Merck Co. and has been widely used in the U.S. and other countries since the early 1970s. Partial sequencing has recently shown that the vaccine contains a mixture of two substrains with substantially different nucleotide sequences. We have determined the complete genomic sequences of both substrains and identified 414 nucleotide differences (2.69%), leading to 87 amino acid substitutions (1.67%). We used this information to develop methods for quantification of the substrain components in vaccine samples based on PCR and restriction enzyme cleavage and oligonucleotide microarray hybridization and monitored their dynamics in viral populations propagated in different conditions. Passaging Jeryl Lynn strain in Vero or CEF cell cultures resulted in rapid selection of the major component JL1, while growth in embryonated chicken eggs (ECE) favored accumulation of the minor component JL2. Based on the findings presented here, it is proposed that the substrain composition of Jeryl Lynn vaccine can be monitored as a part of its quality control to ensure consistency of the vaccine.