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Karen Thudium,1 Jorge Gallo,2 Emmanuel Bouillaud,2 Carolin Sachs,2 Simantini Eddy,1 Wing Cheung1 1Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA; 2Novartis Pharma AG, Basel, Switzerland Background: The mammalian target of rapamycin (mTOR) inhibitor everolimus has a well-established pharmacokinetics profile. We conducted a randomized, single-center, open-label, two-sequence, two-period crossover study of healthy volunteers to assess the relative bioavailability of everolimus administered as one 5 mg tablet or five 1 mg tablets. Methods: Subjects were randomized 1:1 to receive everolimus dosed as one 5 mg tablet or as five 1 mg tablets on day 1, followed by a washout period on days 8–14 and then the opposite formulation on day 15. Blood sampling for pharmacokinetic evaluation was performed at prespecified time points, with 17 samples taken for each treatment period. Primary variables for evaluation of relative bioavailability were area under the concentration–time curve from time zero to infinity (AUCinf) and maximum blood concentration (Cmax). Safety was assessed by reporting the incidence of adverse events (AEs). Results: Twenty-two participants received everolimus as one 5 mg tablet followed by five 1 mg tablets (n=11) or the opposite sequence (n=11). The Cmax of five 1 mg tablets was 48% higher than that of one 5 mg tablet (geometric mean ratio, 1.48; 90% confidence interval [CI], 1.35–1.62). AUCinf was similar (geometric mean ratio, 1.08; 90% CI, 1.02–1.16), as were the extent of absorption and the distribution and elimination kinetics. AEs, all grade 1 or 2, were observed in 54.5% of subjects. Conclusion: Although the extent of absorption was similar, the Cmax of five 1 mg tablets was higher than that of one 5 mg tablet, suggesting these formulations lead to different peak blood concentrations and are not interchangeable at the dose tested. Keywords: absorption kinetics, healthy volunteers

Putative envelope glycoproteins, E1(gp33) and E2(gp72), copurified from HeLa cells infected with a recombinant vaccinia virus expression vector, were used to immunize seven chimpanzees. All vaccines exhibited a powerful humoral immune response. Total protection against an i.v. challenge with homologous hepatitis 6 virus 1 was evident in the five highest responders.

Novel therapeutic approaches combining immune-checkpoint inhibitors are needed to improve clinical outcomes for patients with cancer. Lymphocyte-activation gene 3 (LAG-3) is an immune-checkpoint molecule that inhibits T-cell activity and antitumor immune responses, acting through an independent mechanism from that of programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Here, we describe the development and preclinical characterization of relatlimab, a human antibody that binds to human LAG-3 with high affinity and specificity to block the interaction of LAG-3 with the ligands MHC II and fibrinogen-like protein-1, and to reverse LAG-3-mediated inhibition of T-cell function in vitro. Consistent with previous reports, in mouse models, the combined blockade of LAG-3 and PD-1 with surrogate antibodies resulted in enhanced antitumor activity greater than the individual blockade of either receptor. In toxicity studies in cynomolgus monkeys, relatlimab was generally well tolerated when combined with nivolumab. These results are consistent with findings from the RELATIVITY-047 phase II/III trial showing that relatlimab combined with nivolumab is a well-tolerated regimen that demonstrates superior progression-free survival compared with nivolumab monotherapy in patients with unresectable or metastatic melanoma., (©2022 The Authors; Published by the American Association for Cancer Research.)

Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.

Purpose: The success of checkpoint blockade has led to a significant increase in the development of a broad range of immunomodulatory molecules for the treatment of cancer, including agonists against T-cell costimulatory receptors, such as OX40. Unlike checkpoint blockade, where complete and sustained receptor saturation may be required for maximal activity, the optimal dosing regimen and receptor occupancy for agonist agents is less well understood and requires further study., Experimental Design: We integrated both preclinical and clinical biomarker data sets centered on dose, exposure, receptor occupancy, receptor engagement, and downstream pharmacodynamic changes to model the optimal dose and schedule for the OX40 agonist antibody BMS-986178 alone and in combination with checkpoint blockade., Results: Administration of the ligand-blocking anti-mouse surrogate antibody OX40.23 or BMS-986178 as monotherapy or in combination with checkpoint blockade led to increased peripheral CD4 + and CD8 + T-cell activation in tumor-bearing mice and patients with solid tumors, respectively. OX40 receptor occupancy between 20% and 50% both in vitro and in vivo was associated with maximal enhancement of T-cell effector function by anti-OX40 treatment, whereas a receptor occupancy > 40% led to a profound loss in OX40 receptor expression, with clear implications for availability for repeat dosing., Conclusions: Our results highlight the value of an integrated translational approach applied during early clinical development to aggregate preclinical and clinical data in an effort to define the optimal dose and schedule for T-cell agonists in the clinic., (©2019 American Association for Cancer Research.)

The monoclonal antibodies ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) have shown remarkable antitumor activity in an increasing number of cancers. When combined, ipilimumab and nivolumab have demonstrated superior activity in patients with metastatic melanoma (CHECKMATE-067). Here we describe the preclinical development strategy that predicted these clinical results. Synergistic antitumor activity in mouse MC38 and CT26 colorectal tumor models was observed with concurrent, but not sequential CTLA-4 and PD-1 blockade. Significant antitumor activity was maintained using a fixed dose of anti-CTLA-4 antibody with decreasing doses of anti-PD-1 antibody in the MC38 model. Immunohistochemical and flow cytometric analyses confirmed that CD3+ T cells accumulated at the tumor margin and infiltrated the tumor mass in response to the combination therapy, resulting in favorable effector and regulatory T-cell ratios, increased pro-inflammatory cytokine secretion, and activation of tumor-specific T cells. Similarly, in vitro studies with combined ipilimumab and nivolumab showed enhanced cytokine secretion in superantigen stimulation of human peripheral blood lymphocytes and in mixed lymphocyte response assays. In a cynomolgus macaque toxicology study, dose-dependent immune-related gastrointestinal inflammation was observed with the combination therapy; this response had not been observed in previous single agent cynomolgus studies. Together, these in vitro assays and in vivo models comprise a preclinical strategy for the identification and development of highly effective antitumor combination immunotherapies., Competing Interests: The studies described in this manuscript were sponsored by Bristol-Myers Squibb. The funder provided support in the form of salaries for all authors (MJS JJE RJJ LL MH KT DY MQ JV CW BC PMC DB AJK) and was involved in study design, data collection and analysis, decision to publish, and preparation of the manuscript. The specific roles of these authors are articulated in the Authors’ Contributions section. In addition, the funder provided support in the form of salaries to individuals listed in the Acknowledgements section (LW MS IC CB).

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response., (© 2011 Blackwell Publishing Ltd.)

Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55(Gag) antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55(gag) replicon RNA packaged within SIN envelope glycoproteins and SIN-p55(gag) replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8(+) T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.

DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.

The 3C proteinase from the hepatitis A virus (HAV) was cloned into a multicopy expression vector in Escherichia coli under control of the tac promoter. The resulting plasmid construction produced 3C proteinase as a soluble and active enzyme constituting approximately 10% of total cellular proteins. The enzyme was purified to apparent homogeneity as judged by SDS gel electrophoresis and HPLC reversed-phase and FPLC ion-exchange chromatography. A colorimetric assay was developed, and synthetic peptides derived from the predicted cleavage sites of the HAV polyprotein were tested for proteolysis of the enzyme. The peptide representing the 2B/2C cleavage site was cleaved most efficiently with a Km and kcat of 2.1 +/- 0.5 mM and 1.8 +/- 0.1 s-1, respectively. Site-directed mutagenesis was then used to identify the cysteine at position 172 as the active site nucleophile. Finally, the purified enzyme showed the expected endoproteinase activity on the P1 precursor protein generated by in vitro transcription/translation.