Babraham Bioscience Technologies (United Kingdom)

Vaccines are central to the strategy to control antimicrobial resistant (AMR) bacterial infections; one multidrug resistant pathogen of particular concern is Acinetobacter baumannii. In this study we identified two novel A. baumannii antigens using mass spectrometry and phage expression: Oxa23 and PAL. These genes are highly conserved between different isolates of A. baumannii and recognised by convalescent human sera. We explored their protective immunity using two different vaccine platforms, recombinant outer membrane vesicles (rOMV) and mRNA. RNA vaccine immunised mice had significantly reduced bacterial load in their lower airways following challenge with carbapenem resistant A. baumannii, with Oxa23 providing better protection than PAL. We then compared routes of delivery and RNA vaccine platforms, demonstrating that intranasally delivery of mRNA encoding OXA-23 (formulated with GL67A) significantly reduced disease severity and enhanced bacterial clearance. These studies validate in silico identified antigens through challenge studies and novel mucosal vaccine delivery approaches.

Abstract BackgroundTauopathies, such as frontotemporal dementia (FTD) and Alzheimer’s dimentia (AD), are neurodegenerative diseases characterized by the pathological aggregation of paired helical filaments (PHFs) or neurofibrillary tangles (NFTs) within neurons and glia, leading to cell death1. PHFs and NFTs are formed by aggregation of hyperphosphorylated tau1. Mutations in the microtubule‐associated protein tau (MAPT) gene result in tauopathies. Moreover, FTD is a common clinical syndrome of 4 repeat (4R) tauopathies, as defined by aggregation of tau protein isoforms with four microtubule binding domains2. Here, we aimed to develop and characterize a physiologically relevant and robust in vitro FTD model, to aid the future development of FTD disease therapeutics.MethodUsing CRISPR‐Cas9 gene editing technology, familial mutations MAPT P301S and N279K underlying AD/FTD3 were engineered into an iPSC line that carries the opti‐oxTM technology and can rapidly be reprogrammed into glutamatergic neurons4. By means of immunocytochemistry we characterized b‐amyloid oligomer treated neurons, and neurons with the MAPT mutations to assess tau hyperphosphorylation.ResultThe CRISPR‐edited MAPT neurons can mature and show a healthy morphology. b‐amyloid oligomers (dose dependently) induce tau hyperphosphorylation in wild type glutamatergic neurons. Total tau expression is reduced in MAPT P301S homozygous and N279K heterozygous cells when compared to wild type cells. The MAPT P301S cells also show hyperphosphorylation for p‐tau 217, p‐tau202/205, and p‐tau404, and the N279K cells show hyperphosphorylation for p‐tau202/205, and p‐tau404.ConclusionThe elevated p‐tau to total tau ratio measured by the immunocytochemistry assay indicates the potential of at least two cell lines as possible disease models to aid future research into developing AD/FTD disease therapeutics.

Multiple tumor types overexpress Nectin-4 and the antibody–drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a “Bicycle Toxin Conjugate” (BTC) consisting of a Nectin-4–binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1–2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.

A practically useful coupling reaction between aromatic halides and redox-active esters was realized by nickel catalysis through the use of a packed zinc bed column in continuous flow. Multiple reuse of the column showed a negligible decrease in efficiency, affording high space/time yields. A wide range of substrates, including a number of heteroaryl halides and polyfunctional materials were coupled in generally good yields. Longer-time and larger-scale experiments further demonstrates the robustness of the system.

In response to Colin Pykett's Lateral Thoughts article "My metronomes won't synchronize" (January p56).

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.