AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Modulating Akt signalling to enhance CAR T-cell therapy

CD19 targeting chimeric antigen receptor (CAR) T-cells deliver excellent clinical responses in relapsed/refractory acute lymphoblastic leukaemia (B-ALL) and high-grade B cell lymphoma. However, many patients do not respond or relapse post treatment because of poor CAR T-cell expansion and persistence. Adoptive cell transfer studies have shown that stem-cell memory/naïve/central memory (Tscm/Tn/Tcm) T-cell subsets compared to effector/terminal (Te/Tte) subsets, deliver superior T-cell expansion, persistence, and anti-tumour efficacy in-vitro and in-vivo. In patients, the proportion of Tn/Tcm subsets can vary considerably, and current manufacturing protocols prioritising mass cell expansion often give rise to Te/Tte skewed products. Studies have demonstrated that the ex-vivo inhibition of Akt signalling, an important mediator of T-cell activation and differentiation, can enrich such Tn/Tcm populations. A novel CD19 targeting single chain fragment variable (scFv) was developed at UCL and named CAT. The CAT scFv was found to have lower affinity for the CD19 target over the FMC63 scFv, utilised in current FDA approved KymriahTM, YescartaTM and TecartusTM CAR therapies. This CAT CAR has demonstrated durable responses against paediatric/adult B-ALL and was used as a model to explore the effects of ex-vivo Akt inhibition. We describe a genetic engineering approach of Akt inhibition through the expression of a dominant negative Akt1 molecule and highlight the benefits of pharmacological Akt inhibition. We assessed the incorporation of Akt inhibitor VIII (further referred to as VIII), originally developed by Merck Research Laboratories, Pennsylvania, USA (Compound 16g) into the CliniMACS Prodigy® manufacture process, utilised in current UCL based CAR T-cell trials, in both heathy/B-ALL patients at small/large scale. We show that ex-vivo Akt inhibition can promote enrichment of less differentiated Tscm/Tcm subsets with improved expansion and cytotoxicity against antigen expressing targets in-vitro and superior anti-tumour activity in-vivo. Inhibition using VIII supported enrichment of CD4 Th1 and Th17 subsets with no skew towards Th2 and Tregs, as previously reported resulting in increased T-cell polyfunctionality. Transcriptome and metabolic analysis revealed a signature for autophagy in CAR CD8 T-cells validated by an increase in autophagic vesicles, not previously described in the context of ex-vivo Akt inhibition in CAR T-cells. VIII-treated CAR CD8 T-cells were found to have a unique metabolic profile with high mitochondrial membrane potential (ΔΨm) and increased fatty acid oxidation to support energy demands of enhanced expansion and cytotoxicity. Lastly, we demonstrate that Akt inhibition can be incorporated into large scale automated manufacturing processes with the potential to generate functionally superior products compared to standard untreated products. This ultimately may help to improve clinical responses by rescuing patients/products at risk of CD19+ relapse

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

Background AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets.Methods VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale.Results AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP.Conclusion Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Bone marrow mesenchymal stem cells do not enhance intra-synovial tendon healing despite engraftment and homing to niches within the synovium

BackgroundIntra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs.MethodsA lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1-2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1-2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing.ResultsThe MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination.ConclusionsUnlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair

Durable responses and low toxicity after fast off-rate cd19 chimeric antigen receptor-t therapy in adults with relapsed or refractory b-cell acute lymphoblastic leukemia

PURPOSE: Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS: Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS: Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease–negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4%-84.4%) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION: AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL