Background Clonal neoantigens are formed early in cancer evolution and have been identified as a subset of patient specific mutations that are associated with improved clinical benefit, representing great promise as targets for next generation T cell therapies. Cell therapies targeting multiple clonal neoantigens represent a unique personalised approach to treating solid cancer, as they are present on all cancer cells, minimising the risk of tumour escape, and absent from healthy tissue. Process 2 of the VELOSTM manufacturing platform has successfully demonstrated the feasibility of generating clonal neoantigen-reactive T cell (cNeT) products for the treatment of advanced NSCLC, (NCT04032847) and melanoma (NCT03997474) in two first-in-human studies. Here we demonstrate that implementation of an optimised VELOSTM platform (Process 2b) in clinical manufacturing can generate a significant dose boost of highly potent and reactive CD8+ and CD4+ cNeT for clinical use compared to both Process 1 and Process 2. Method Briefly, tumour-infiltrating lymphocytes (TIL) were isolated from tumour fragments and Dendritic Cells (DCs) generated from whole blood, prior to cryopreservation. Patient-specific clonal neoantigens were predicted using our proprietary PELEUSTM bioinformatic platform, enabling manufacture of synthetic peptides for each patient. The co-culture of TIL and peptide loaded DCs allows the selective expansion of cNeT, prior to a polyclonal T cell boost step. Results Here we present clinical manufacturing data on 21 tumour samples (13 NSCLC and 8 melanoma) generated with Process 2b and demonstrate a 5-fold increase in the median CD3+ TIL yield compared to 33 TIL intermediates (13 NSCLC and 20 melanoma) generated with Process 2 (30M vs. 147M). Improvements in TIL yield at the pre-expansion step have driven a 10-fold increase in the median cNeT dose (17M vs. 167M) following co-culture in 15 clinical batches manufactured with VELOSTM Process 2b (14 – 6,347M) compared to 17 batches manufactured with Process 2 (1.4 – 6,409M). Median clonal reactivity was 16% (0.2 – 96%) with products manufactured with Process 2b compared to 10% (0 – 97.6%) with Process 2. Furthermore, peptide deconvolution identified products with multiple single T cell reactivities to clonal neoantigens demonstrating a highly polyclonal product (mean number of unique variants: 7, range 0 – 90). Conclusions We demonstrate that optimised VELOSTM Process 2b incorporating the PELEUSTM platform for prediction of clonal neoantigens can generate significantly higher cNeT doses and in some cases >1B in concert with accurately identifying the active drug component for the treatment of advanced NSCLC and melanoma.

Regulatory T cells (Tregs) are a lymphocyte subset with intrinsic immunosuppressive properties that can be expanded in large numbers ex vivo and have been shown to prevent allograft rejection and promote tolerance in animal models. To investigate the safety, applicability, and biological activity of autologous Treg adoptive transfer in humans, we conducted an open-label, dose-escalation, Phase I clinical trial in liver transplantation. Patients were enrolled while awaiting liver transplantation or 6-12 months posttransplant. Circulating Tregs were isolated from blood or leukapheresis, expanded under good manufacturing practices (GMP) conditions, and administered intravenously at either 0.5-1 million Tregs/kg or 3-4.5 million Tregs/kg. The primary endpoint was the rate of dose- limiting toxicities occurring within 4 weeks of infusion. The applicability of the clinical protocol was poor unless patient recruitment was deferred until 6-12 months posttransplant. Thus, only 3 of the 17 patients who consented while awaiting liver transplantation were dosed. In contrast, all six patients who consented 6-12 months posttransplant received the cell infusion. Treg transfer was safe, transiently increased the pool of circulating Tregs and reduced anti-donor T cell responses. Our study opens the door to employing Treg immunotherapy to facilitate the reduction or complete discontinuation of immunosuppression following liver transplantation.

Background: Clonal neoantigens are formed early in cancer evolution and have been identified as a subset of patient specific mutations that are associated with improved clinical benefit and represent great promise as targets for the next generation of T cell therapies. Developing T cell therapies that target multiple clonal neoantigens represents a unique personalized approach to treating solid cancer, as they are present on all cancer cells, minimizing the risk of tumour escape, and absent from healthy tissue, potentially eliminating off-target toxicities. Access to sequencing data from over 600 NSCLC patients enrolled in the UK TRACERx study has enabled the development of the Achilles PELEUSTM bioinformatic platform. By opening an ethically approved tissue collection study NCT03517917, enabling access to matched tumour and blood samples from patients with selected cancers, our clonal neoantigen reactive T cell (cNeT) manufacturing process and supply chain has been validated for use in clinical trials. Methods: Matched tumor and blood samples were procured at the time of routine surgery from ten patients (eight with newly diagnosed stage I-III NSCLC and two with metastatic melanoma) for at-scale GMP runs. Briefly, TIL were isolated from tumor fragments and immature dendritic cells (DCs) generated from whole blood, prior to cryopreservation as intermediate products. Patient-specific clonal neoantigens were predicted using our proprietary PELEUSTM bioinformatic platform, enabling the manufacture of synthetic peptide masterpools to be used for the enrichment of cNeT in the VELOSTM manufacturing process. Co-culture of pre-expanded TIL and patient DCs loaded with clonal neoantigen peptides drives the selective expansion of cNeT, eliminating the requirement for high non-physiological levels of IL-2. Results: Here we present the successful scaled GMP production of cNeT from both primary and metastatic tumors using the VELOSTM manufacturing process in ten patients. All final products met QC release criteria and were composed of both CD4+ and CD8+ T cells. Extensive characterization of T cell responses showed cNeT exhibited functional responses determined by cytokine secretion following re-challenge, and specificity in response to clonal neoantigen peptides. Peptide deconvolution of masterpools identified multiple single T cell clone reactivities to clonal neoantigens in the final product. Conclusions: The VELOSTM process incorporating the PELEUSTM bioinformatic platform for prediction of clonal neoantigens is a novel platform for generating personalized T cell products directed at multiple cancer clonal neoantigen targets and has the potential to be utilized across a variety of solid tumors. This study demonstrates the feasibility of generating cNeT for the treatment of both advanced NSCLC and recurrent or metastatic melanoma and supported the successful regulatory approval in two first-in-human studies (NCT04032847 and NCT03997474) which opened in the UK in 2019. Citation Format: Henrieta Fraser, Rebecca Pike, Sarah Thirkell, Asiya Arshad, Sam Jide-Banwo, Hollie Bartley, Evi Rologi, Michal Pruchniak, Shreenal Patel, Jennine Mootien, Jane Robertson, Andrew Craig, Max Salm, Katy Newton, Luke Goodsell, Fong Chan, Gareth Wilson, Stephen Frenk, Iraj Ali, Karl Peggs, Mark W. Lowdell, Lyra Del Rosio, Andrew Hayes, Samra Turajlic, Farah Islam, David Lawrence, Mariam Jamal-Hanjani, Martin D. Forster, Edward Samuel. The development of a personalized autologous clonal neoantigen T cell therapy for the treatment of solid cancer using the VELOSTM manufacturing platform generates highly potent and reactive CD8+ and CD4+ T cells for clinical use [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT054.

Solid organ transplantation is the treatment of choice for patients with end-stage organ dysfunction. Despite improvements in short-term outcome, long-term outcome is suboptimal due to the increased morbidity and mortality associated with the toxicity of immunosuppressive regimens and chronic rejection (1-5). As such, the attention of the transplant community has focused on the development of novel therapeutic strategies to achieve allograft tolerance, a state whereby the immune system of the recipient can be re-educated to accept the allograft, averting the need for long-term immunosuppression. Indeed, reports of "operational" tolerance, whereby the recipient is off all immunosuppressive drugs and maintaining good graft function, is well documented in the literature for both liver and kidney transplantations (6-8). However, this phenomenon is rare and in the setting of liver transplantation has been shown to occur late after transplantation, with the majority of patients maintained on life-long immunosupression to prevent allograft rejection (9). As such, significant research has focused on immune regulation in the context of organ transplantation with regulatory T cells (Tregs) identified as cells holding considerable promise in this endeavor. This review will provide a brief introduction to human Tregs, their phenotypic and functional characterization and focuses on our experience to date at the clinical translation of Treg immunotherapy in the setting of solid organ transplantation.

The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, while maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicenter phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial, ThRIL, recently commenced at King's College London, proposes to use Treg cell therapy to induce tolerance in liver transplant recipients, the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads, IL-2 and rapamycin. We report the enrichment of a pure, stable population of Tregs (>95% CD4(+)CD25(+)FOXP3(+)), reaching adequate numbers for their clinical application. Our protocol proved successful in, influencing the expansion of superior functional Tregs, as compared to freshly isolated cells, whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF), a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL, will undoubtedly inform the improved management of the liver transplant recipient.