Preclinical development of a vaccine-based immunotherapy regimen (VBIR) that induces potent and durable T cell responses to tumor-associated self-antigens.

The development of therapeutic cancer vaccines remains an active area, although previous approaches have yielded disappointing results. We have built on lessons from previous cancer vaccine approaches and immune checkpoint inhibitor research to develop VBIR, a vaccine-based immunotherapy regimen. Assessment of various technologies led to selection of a heterologous vaccine using chimpanzee adenovirus (AdC68) for priming followed by boosts with electroporation of DNA plasmid to deliver T cell antigens to the immune system. We found that priming with AdC68 rapidly activates and expands antigen-specific T cells and does not encounter pre-existing immunity as occurs with the use of a human adenovirus vaccine. The AdC68 vector does, however, induce new anti-virus immune responses, limiting its use for boosting. To circumvent this, boosting with DNA encoding the same antigens can be done repetitively to augment and maintain vaccine responses. Using mouse and monkey models, we found that the activation of both CD4 and CD8 T cells was amplified by combination with anti-CTLA-4 and anti-PD-1 antibodies. These antibodies were administered subcutaneously to target their distribution to vaccination sites and to reduce systemic exposure which may improve their safety. VBIR can break tolerance and activate T cells recognizing tumor-associated self-antigens. This activation lasts more than a year after completing treatment in monkeys, and inhibits tumor growth to a greater degree than is observed using the individual components in mouse cancer models. These results have encouraged the testing of this combination regimen in cancer patients with the aim of increasing responses beyond current therapies.

Oncolytic Vaccinia Virus Gene Modification and Cytokine Expression Effects on Tumor Infection, Immune Response, and Killing.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial phenylalanyl-tRNA synthetase.

A series of benzyl phenyl ethers (BPEs) is described that displays potent inhibition of bacterial phenylalanyl-tRNA synthetase. The synthesis, SAR, and select ADMET data are provided.

Antibacterial oxazolidinones possessing a novel C-5 side chain. (5R)-trans-3-[3-Fluoro-4- (1-oxotetrahydrothiopyran-4-yl)phenyl]-2- oxooxazolidine-5-carboxylic acid amide (PF-00422602), a new lead compound.

Oxazolidinones possessing a C-5 carboxamide functionality (reverse amides) represent a new series of compounds that block bacterial protein synthesis. These reverse amides also exhibited less potency against monoamine oxidase (MAO) enzymes and thus possess less potential for the side effects associated with MAO inhibition. The title compound (14) showed reduced in vivo myelotoxicity compared to linezolid in a 14-day safety study in rats, potent in vivo efficacy in murine systemic infection models, and excellent pharmacokinetic properties.

Simultaneous screening of multiple bacterial tRNA synthetases using an Escherichia coli S30-based transcription and translation assay.

The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.

A distal methyl substituent attenuates mitochondrial protein synthesis inhibition in oxazolidinone antibacterials.

Oxazolidinone analogs bearing substituted piperidine or azetidine C-rings are described. Analogs with a methyl group at the 3-position of the azetidine ring or the 4-position of the piperidine ring exhibited reduced mitochondrial protein synthesis inhibition while retaining good antibacterial potency.

Synthesis and SAR of novel conformationally-restricted oxazolidinones possessing gram-positive and fastidious gram-negative antibacterial activity. Part 1: Substituted pyrazoles.

A novel series of conformationally-restricted oxazolidinones was synthesized which possess a fused pyrazole ring substituted with various alkyl, aryl and heteroaryl substituents. A number of analogs exhibited potent activity against both gram-positive and fastidious gram-negative organisms.

Synthesis and SAR of novel conformationally restricted oxazolidinones possessing gram-positive and fastidious gram-negative antibacterial activity. Part 2: Amino substitutions on heterocyclic D-ring system.

A novel series of conformationally restricted oxazolidinones was synthesized, in which the heterocyclic D ring was substituted with various amino groups. Several analogs exhibited potent activity against both gram-positive and fastidious gram-negative organisms. Certain amino-substituted analogs also exhibited improved aqueous solubility compared to the corresponding un-substituted heterocyclic D-ring analogs.

Synthesis and structure-activity studies of novel benzocycloheptanone oxazolidinone antibacterial agents.

We describe a novel class of benzocycloheptanone derived oxazolidinone antibacterial agents. The synthesis and antibacterial activities with structure variation is discussed.