Concept Life Sciences (United Kingdom)

As a part of the ongoing COVID Moonshot project, an expedient approach to 1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylates is described. The method is based on a one-pot tandem Michael amination – lactamization sequence starting from of 2-(3-alkoxymethoxy-3-oxoprop-1-en-2-yl)benzoic acids and primary amines. The scope of the N-nucleophiles included various aliphatic, and aromatic, and heteroaromatic primary amines; the reaction tolerates and was compatible with ether, ester, amide, alcohol, carbamate, and free alcoholazole, or azine moieties. The developed protocol; allowed for the preparation of target tetrahydroisoqunoline derivatives in 40–75% yield on up to 109 g scale. A mechanistic scheme involving a ring-chain tautomerism for the electrophilic component of the reaction is proposed. The method was used in the synthesis of DNDI-6510, SARS-CoV-2 main protease inhibitor that showed promising results obtained in preclinical studies.

Abstract Fibroblast activation protein α (FAP) is a post-proline protease and solid-tumor target which Avacta’s pre|CISION® peptide moiety linker selectively uses as a differentiated means to target the tumor microenvironment (TME). The potential of the pre|CISION® technology is highlighted by the promising clinical data from AVA6000, a pre|CISION® enabled Doxorubicin peptide drug conjugate. FAP expression is tightly regulated in normal adult tissues, with low to undetectable levels in most healthy cells. However, it becomes highly upregulated in the TME where it is expressed by cancer-associated fibroblasts (CAFs). This selective expression pattern has been observed in over 90% of epithelial tumors, including breast, colorectal, pancreatic, lung, brain, and ovarian cancer. We implemented a multi-faceted approach to further the understanding of FAP biology and the potential application for pre|CISION® medicines across a broad range of tumors. We conducted a comprehensive analysis of FAP using real-world data from Tempus AI’s LENS database encompassing over 140, 000 tumor samples across 30+ solid tumor types. We evaluated FAP pan-cancer RNA expression, spatial organization of FAP in the TME, as well as correlations with biomarkers. We further analyzed FAP expression pre- and post-treatment and performed in-depth analysis to evaluate FAP expression in tumor subtypes and primary or metastatic lesions. Single-cell RNA-sequencing (scRNA-seq) analysis provided insights into FAP-expressing cell populations as well as correlations between CAF abundance and FAP. FAP expression was evaluated at the protein level using IHC analysis across multiple tumor indications. Multi-immunofluorescence IHC (multi-IF) was used to evaluate the spatial localization of FAP relative to tumor cells and vasculature. Our findings reveal high FAP expression across a range of tumor indications and demonstrate a correlation with novel biomarkers of interest. IHC analysis confirms a direct relationship between FAP RNA and protein expression, while multi-IF imaging and concentric partitioning analysis for a range of solid tumor indications shows the spatial association of tumor cells, blood vessels and CAFs expressing FAP. ScRNA-seq data confirms high level FAP expression on fibroblasts in stromal-rich tumors. Finally, co-culture models were established with tumor cells and matched fibroblasts which represent tumors with differing levels of FAP enzyme activity. Avacta’s preCISION® peptide-drug conjugates elicit bystander cell kill over a range of physiological FAP levels within these co-culture models. Combined, these analyses emphasize the significance of FAP as an important stromal target for focused delivery of targeted oncology therapeutics, and guide further development of the preCISION® pipeline. Citation Format: Ruairidh Edwards, 1 Curtis Rink, 1 Giulia Tagliavini, 2 Christopher Mills, 2 David Jones, 1 Michelle Morrow1. Investigating fibroblast activation protein alpha (FAPα) as a therapeutic target for delivery of pre|CISION® cancer medicines: Expression, spatial localization and functional insights [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2699.

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4+ T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.

BackgroundAn important facet of the hostile tumor microenvironment (TME) and a recognized driver of cancer immunosuppression is extracellular acidification which results from the glycolytic nature of rapidly proliferating cancer cells. Pathios identified GPR65, a pH-sensing G protein-coupled receptor (GPCR), as the primary sensor of this acidity in human immune cells. Activation of GPR65 is particularly pronounced in macrophages where it leads to the downregulation of key chemokines and anti-tumorigenic cytokines, and the upregulation of pro-tumorigenic wound repair and remodeling factors. In dendritic cells GPR65 signaling suppresses antigen presentation pathways, whereas in T cells and natural killer cells it leads to the suppression of chemokine receptors required for effective tumor homing. Consequently, acid sensing by GPR65 can explain tumor resistance to current immunotherapies. Validating the relevance of this target in immuno-oncology, cancer patients homozygous for the hypomorphic GPR65 variant (I231L) exhibit profoundly improved survival compared to patients with other genotypes.MethodsPathios has developed PTT-4256, a first-in-class small molecule inhibitor of GPR65. The in vitro efficacy of PTT-4256 under low pH conditions was determined by cAMP screening, gene expression and cytokine release profiling of primary immune populations. Anti-tumor activity was assessed using the MC38 and B16.F10 syngeneic mouse models, whilst immunomodulatory effects in MC38 tumor-infiltrating leukocytes were profiled by targeted RNAseq. Notably, PTT-4256 is markedly more potent at human GPR65 versus the mouse receptor. To determine its full potential in patients and to develop an accurate prediction of efficacious human exposure, we employed a range of approaches including molecular pharmacology, site-directed mutagenesis, PK/PD modelling, and genetically engineered mice.ResultsPTT-4256 fully counteracts the low pH-driven immunosuppressive transcriptional program in human and murine immune cells and restores the secretion of key pro-inflammatory cytokines and chemokines in macrophages. PTT-4256 displays an excellent oral PK profile across species and elicits significant monotherapy efficacy in MC38 and B16.F10 mouse models. Through molecular modelling and site-directed mutagenesis we identified three key residues that explained the difference in potency of PTT-4256 between mouse and human GPR65. Incorporating these residues in a transgenic knock-in mouse fully restored human pharmacology in vitro and provided an important translational platform.ConclusionsGPR65 is a key checkpoint on tumor-infiltrating immune cells that links the chronically acidic TME to tumor-promoting immunosuppression. The GPR65 inhibitor PTT-4256 restores anti-tumor immunity and demonstrates pronounced single-agent efficacy in mice, and is therefore being advanced towards clinical development.Ethics ApprovalProtocols or procedures involving the care and use of animals in studies in China were reviewed and approved by the Institutional Animal Care and Use Committee of Crown Bioscience. During studies, the care and use of animals was conducted in accordance with the regulation of the Association for Assessment and Accreditation of Laboratory Animal Care.Studies involving the welfare and use of animals within the UK complied with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and Council of 22/September/2010 on the protection of animals used for scientific purposes and UK Home Office guidance on the implementation of the Act and applicable codes of practice for the care and housing of laboratory animals.

BackgroundEMT is the process whereby epithelial cells acquire mesenchymal characteristics. This process is highly regulated and required in development and tissue regeneration. However, in cancer the process becomes dysregulated leading to increased invasion, metastasis and tumor progression. Macrophages are one of the major infiltrating immune cells in the tumor microenvironment and have been proposed as a primary driver of pathological EMT through interactions with tumour cells. EMT and macrophage infiltration have been correlated to enhance tumor progression in multiple cancer types, making it a potential therapeutic target. In this study we generate and characterise a series of human macrophage subtypes via cytokine polarisation and show that multiple macrophage polarised states enhance the EMT phenotype.MethodsHuman macrophages were differentiated using GM-CSF or M-CSF prior to polarisation with individual cytokines (M-CSF + IFN-g; M-CSF + IL-4; M-CSF + IL-1b; M-CSF + IL-10; M-CSF + IL-6) to generate different putative macrophage phenotypes (M1, M2a, M2b, M2c, M2d) as evidenced by differences in cytokine secretion, gene transcription and protein expression. Epithelial cells were either stimulated with macrophage conditioned medium or in co-culture with macrophages in a scratch wound assay (Incucyte) and analysed by immunocytochemistry (Vimentin, SNAIL, E-cadherin). Supernatants of the stimulated macrophages were assessed by Luminex bead array and the macrophages subtypes were also assessed using bulk RNA sequencing for differences in transcriptome.ResultsSupernatants derived from macrophages and macrophage co-culture were shown to drive EMT, both in migratory capacity and EMT associated protein expression. Our results demonstrate that even a single cytokine can alter the polarisation and subsequent cytokine production of the macrophage; with IL-12 and TNF-α associated with more pro-inflammatory phenotypes (M1), and IL-10 and VEGFA more anti-inflammatory or tissue remodelling phenotypes (M2). Inhibition of the TGF-βRI by galunisertib can partially reverse the induction of EMT migration and protein expression.ConclusionsThe data shows that macrophages are plastic and the cytokines they produce can directly drive the EMT process in vitro via multiple mechanisms. This supports the conclusion that individual cytokine inhibition such as TGF-βRI alone is likely to be ineffective as macrophages produce multiple factors that can induce EMT. Using this powerful model, researchers can gain better understanding of complex interactions occurring in the tumor microenvironment, identify molecular targets with potential to block cancer cell metastasis and evaluate candidate drug efficacy.

Nanoparticle-assisted enhanced oil recovery (Nano-EOR)has attractedintensive interest in the laboratory as a promising oil recovery technology.However, the nanoparticles’ stability and long-distance deliveryof nanoparticles (NPs) in large-scale reservoirs are two main challenges.In this work, we developed a novel concept of in situ synthesizing NPs at the oil–water interface inside the reservoirfor EOR instead of injecting presynthesized NPs from outside. Thepore-scale flooding experiments show that EOR efficiencies for tertiaryflooding were 6.3% without reaction (Case 3), 14.6% for slow reaction(Case 1), and 25.4% for relatively quick reaction (Case 4). Examinationof the EOR mechanism shows that in situ producedSiO2 NPs in microchannels could alter the substrate wettabilitytoward neutral wetting. Moreover, the produced NPs tended to assembleon the immiscible oil–water interface, forming a barrier towardinterface deformation. As the reaction continued, excessive surface-modifiedNPs could also diffuse into aqueous brine and accumulate as a softgel in the flowing path swept by brine. Collectively, these processesinduced a “shut-off” effect and diverted displacingfluids to unswept areas, which consequently increased the sweep efficiencyand improved the oil recovery efficiency. Auxiliary bulk-scale experimentsalso showed that the reaction-induced nanoparticle synthesis and assemblyat an immiscible interface reduced the interfacial tension and generatedan elastic oil–water interface.

Abstract The induction of CD8+ T cell exhaustion, driven by the tumor microenvironment, is thought to be a major mechanism by which tumors can escape immune surveillance and are able to persist and progress. T cell exhaustion is typically characterized by the expression of co-inhibitory markers such as PD-1, LAG-3 and TIM-3 and a reduced capacity for pro-inflammatory cytokine production. Whilst treatment with therapeutics targeted at checkpoint inhibitors, such as Nivolumab (anti-PD-1), have shown great promise in the treatment of a subset of individuals and tumor types, resistance to these approaches is prevalent. As such, novel approaches are required in order to provide alternative options for the rescue of T cell exhaustion and cancer treatment. Here we describe the characteristics of an in vitro human T cell exhaustion assay whereby in vitro stimulated T cells phenotypically and functionally recapitulate the exhausted T cells found within the tumor microenvironment and where exhaustion can be partially rescued by treatment with anti-PD-1. Furthermore, we demonstrate the effect of treatment with an inhibitor of a novel target identified in exhausted T cells on T cell function; both through analysis of pro-inflammatory cytokine production and the ability to kill tumor cells. These assays and approaches enable investigation into the ability of compounds to influence reversal of T cell exhaustion where anti-PD-1 treatment does not fully reverse the exhausted phenotype and offers the ability to test combination therapy approaches. Citation Format: Rhoanne McPherson, Francis Acklam, Joanne Hay, Mark Barbour, Courtney Grant, Preeti Singh, Adriana Gambardella, Justyna Rzepecka, Stephen Anderton, Hayley Gooding. Implications of Targeting Novel Pathways in Exhausted T Cells for Immuno-Oncology Therapeutic Intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB158.

This paper summarizes the application of the chiral ligand DTBM-SegPHOS in asymmetric catalytic hydrogenation reactions. DTBM-SegPHOS exhibits pronounced catalytic activity and enantioselectivity due to its high electron density and steric bulk. Indeed, it often performs better in reactions in which traditionally used ligands provide poor results. According to the impressive influence of the t-Bu groups of DTBM-SegPHOS and the resulting interactions with various substrates, new substrates bearing t-Bu groups were studied in asymmetric hydrogenations which were not previously possible. The ligand QuinoxP*, which possesses similar structural features to that of DTBM-SegPHOS, was employed for the hydrogenation of such substrates with the best results. The mechanistic studies prove that the unique catalytic behaviors of the complexes of these ligands in asymmetric hydrogenation are a result of the extensive network of weakly attractive interactions between the catalyst and substrate in the transition states. An overview of these impressive ligands in various asymmetric hydrogenations using different transition metal catalysts is provided.