Engineered CAR-T cells targeting the non-functional P2X purinoceptor 7 (P2X7) receptor as a novel treatment for ovarian cancer.

Objectives: Recent studies have identified expression of the non-functional P2X7 (nfP2X7) receptor on various malignant cells including ovarian cancer, but not on normal cells, which makes it a promising tumour-associated antigen candidate for chimeric antigen receptor (CAR)-T-cell immunotherapies. In this study, we assessed the cytotoxic effects of nfP2X7-CAR-T cells on ovarian cancer using in vitro and in vivo models. Methods: We evaluated the effects of nfP2X7-CAR-T cells on ovarian cancer cell lines (SKOV-3, OVCAR3, OVCAR5), normal peritoneal cells (LP-9) and primary serous ovarian cancer cells derived from patient ascites in vitro using monolayer and 3D spheroid assays. We also evaluated the effects of nfP2X7-CAR-T cells on patient-derived tissue explants, which recapitulate an intact tumour microenvironment. In addition, we investigated the effect of nfP2X7-CAR-T cells in vivo using the OVCAR-3 xenograft model in NOD-scid IL2Rγnull (NSG) mice. Results: Our study found that nfP2X7-CAR-T cells were cytotoxic and significantly inhibited survival of OVCAR3, OVCAR5 and primary serous ovarian cancer cells compared with un-transduced CD3+ T cells in vitro. However, no significant effects of nfP2X7-CAR-T cells were observed for SKOV3 or normal peritoneal cells (LP-9) cells with low P2X7 receptor expression. Treatment with nfP2X7-CAR-T cells increased apoptosis compared with un-transduced T cells in patient-derived explants and correlated with CD3 positivity. Treatment with nfP2X7-CAR-T cells significantly reduced OVCAR3 tumour burden in mice compared with un-transduced CD3 cells for 7-8 weeks. Conclusion: This study demonstrates that nfP2X7-CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer.

Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy targeting nfP2X7.

Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel treatment that genetically modifies the patients' own T cells to target and kill malignant cells. However, identification of tumour-specific antigens expressed on multiple solid cancer types, remains a major challenge. P2X purinoceptor 7 (P2X7) is a cell surface expressed ATP gated cation channel, and a dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from multiple tissues, while being undetectable on healthy cells. We present a prototype -human CAR-T construct targeting nfP2X7 showing potential antigen-specific cytotoxicity against twelve solid cancer types (breast, prostate, lung, colorectal, brain and skin). In xenograft mouse models of breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibit robust anti-tumour efficacy. These data indicate that nfP2X7 is a suitable immunotherapy target because of its broad expression on human tumours. CAR-T cells targeting nfP2X7 have potential as a wide-spectrum cancer immunotherapy for solid tumours in humans.

Inertial Microfluidic Purification of CAR-T-Cell Products.

Chimeric antigen receptor T (CAR-T) cell therapy is rapidly becoming a frontline cancer therapy. However, the manufacturing process is time-, labor- and cost-intensive, and it suffers from significant bottlenecks. Many CAR-T products fail to reach the viability release criteria set by regulators for commercial cell therapy products. This results in non-recoupable costs for the manufacturer and is detrimental to patients who may not receive their scheduled treatment or receive out-of-specification suboptimal formulation. It is demonstrated here that inertial microfluidics can, within minutes, efficiently deplete nonviable cells from low-viability CAR-T cell products. The percentage of viable cells increases from 40% (SD ± 0.12) to 71% (SD ± 0.09) for untransduced T cells and from 51% (SD ± 0.12) to 71% (SD ± 0.09) for CAR-T cells, which meets the clinical trials' release parameters. In addition, the processing of CAR-T cells formulated in CryStor yields a 91% reduction in the amount of the cryoprotectant dimethyl sulfoxide. Inertial microfluidic processing has no detrimental effects on the proliferation and cytotoxicity of CAR-T cells. Interestingly, ≈50% of T-regulatory and T-suppressor cells are depleted, suggesting the potential for inertial microfluidic processing to tune the phenotypical composition of T-cell products.

Isolation and identification of actinobacteria from surface-sterilized wheat roots.

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.

Visualization of an endophytic Streptomyces species in wheat seed.

Endophytic filamentous actinobacteria were isolated from surface-sterilized roots of wheat plants. Endophytic colonization of germinating wheat seed was examined using one of these endophytes, Streptomyces sp. strain EN27, tagged with the egfp gene. Endophytic colonization was observed from a very early stage of plant development with colonization of the embryo, endosperm, and emerging radicle.

Complete sequencing and analysis of pEN2701, a novel 13-kb plasmid from an endophytic Streptomyces sp.

A 12,855 bp cryptic plasmid was isolated from strains of an endophytic Streptomyces sp. over a wide geographical area in South Australia. This plasmid was completely sequenced and 13 putative ORFs were identified. Two of the ORFs may be involved in the regulation of host plant genes. ORF7 exhibited homology to a plant transcriptional regulatory protein and ORF1 was a homolog of a plant protein synthesis initiation factor. The plasmid appears to use a novel transfer mechanism for a Streptomyces plasmid. Pocks were detected during conjugative transfer and kor but not tra homologs could be identified. This structure and the sequence of the putative Kor protein are similar to the pFQ series of plasmids isolated from Frankia, another endophytic actinomycete.