IntroductionRetroviral T-cell receptor (TCR) gene transfer is an attractive strategy by which large numbers of antigen-specific T cells can be generated for adoptive transfer. [1][2][3][4] One of the advantages of this technique is that it can be used to circumvent possible impairment of autologous T-cell responses against tumor associated antigens as it bypasses central tolerance. 5 In addition, the introduced TCR specificity can be targeted against poorly immunogenic antigens and high affinity TCR can be selected for transfer. 6 This method has recently seen success in the first clinical trial of TCR gene therapy, where MART1 specific T cells, generated by retroviral gene transfer, were adoptively transferred into patients with metastatic melanoma. The engineered T cells engrafted in 15 of 17 patients, 2 of whom demonstrated long term tumor regression. 7 T cells engineered to express CEA-specific TCR also induced a decrease in CEA levels in 3 patients with metastatic colorectal cancer, and regression of tumor metastases in 1 patient, but were associated with a severe colitis in all 3 patients. 8 The most promising results were seen after adoptive transfer of TCR transduced T cells specific for the NY-ESO-1antigen which resulted in clinical responses in synovial cell sarcoma and in melanoma. 9 As the density of TCR on the surface of cells affects their functional avidity, inefficient TCR expression may impair the success of TCR gene therapy. [10][11][12][13][14] Several different strategies have been developed to increase the expression of introduced ␣ and ␤ chains by reducing the mispairing with endogenous TCR chains. The strategies include replacing the human TCR constant domain with murine sequences, the introduction of an additional disulphide bond into the constant regions, and the production of hybrid molecules consisting of the extracellular portion of TCR chains fused to the intracellular CD3 domain. [15][16][17][18] TCR ␣ and ␤ chains form a complex with 4 invariant CD3 chains: ␥,␦,⑀ and . This complex formation is required in order for the TCR to be expressed on the cell surface, and for signal transduction on antigen recognition. TCR introduced by retroviral gene transfer are likely to be in competition with endogenous TCR molecules for CD3 chains. We used a murine model system to explore whether the co-transfer of TCR genes together with the genes encoding the ␥,␦,⑀ and chains of the CD3 complex can augment TCR expression. Two different TCR, with specificity for Wilms' Tumor antigen 1 (WT1) and influenza nucleoprotein, were used to demonstrate that CD3 is rate limiting for the expression of introduced TCR in gene modified T cells. Co-transduction of CD3 and TCR genes resulted in up to 16-20 fold increase in TCR surface expression and tetramer binding compared with transduction of TCR genes alone. The increase in TCR expression was associated with increased T-cell avidity leading to improved recognition of low concentration of peptide antigen. In vivo TCRϩCD3 co-transduced T cells eradicate tumors fa...

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Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a nonself-viral antigen retained a CD44 low naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor- IntroductionThe Wilms Tumor antigen 1 (WT1) is an attractive target for immunotherapy of leukemia and solid tumors as it is expressed at high levels in many malignancies, whereas progenitor and stem cell populations express only low levels of the WT1 transcription factor. [1][2][3][4][5] In acute myeloid leukemia (AML), high WT1 levels are associated with poor prognosis, and the quantitative measurement of WT1 RNA transcripts is now widely accepted as a sensitive molecular marker for monitoring minimal residual disease in patients undergoing chemotherapy or transplantation. 6 In the past few years, vaccination with WT1 peptides has been tested as a treatment option for various malignancies, including myelodysplasia and leukemia. 7,8 In these studies clinical responses were observed in 60% to 74% of evaluable patients (including stable disease and reduced expression of tumor markers), but correlation with the detection of immunologic responses in peripheral blood was variable. Studies in breast cancer patients showed that WT1-specific T cells were undetectable in peripheral blood, although present in tumor-draining lymph nodes (LNs). 9 Therefore, failure to detect WT1-sepcific T cells in the blood of cancer patients might be the result of selective migration to the site of tumor growth.It is possible that low-level WT1 expression in normal progenitor cells may result in the central deletion of high avidity WT1-specific T cells or induce unresponsiveness by peripheral tolerance mechanisms. However, there is good evidence indicating that central and peripheral tolerance to WT1 is incomplete. First, vaccination in humans can induce self-restricted WT1-specific T-cell responses in some patients, although their frequency is generally low. 10 In addition, WT1-specific T cells were detectable in leukemia patients ...

Background:Patients with acute myeloid leukemia (AML), myelodysplasia (MDS) or tyrosine kinase inhibitor resistant chronic myeloid leukemia (CML) who are unsuitable for consolidative allogeneic stem cell transplantation (alloSCT) have high relapse rates following chemotherapy. Wilms' tumor 1 (WT1) is highly expressed in the majority of acute myeloid leukemias (AML) and in many subtypes of myelodysplasia (MDS) as well as other hematological and solid tumors. WT1 is an intracellular antigen, which makes it difficult to target using current Chimeric Antigen Receptor (CAR)-T cell technologies. The use of genetically modified T cells expressing WT1-specific α/β T cell receptors can re-direct T cell specificity via the recognition of intracellular peptides presented by MHC molecules on the malignant cell surface. Phase I clinical trials of WT1-TCR gene-modified T cells have been conducted in the settings of relapsed disease and post-alloSCT and preliminary data suggests this treatment approach is safe and potentially clinically effective in these cohorts (Tawara et al. Blood. 2017;130(18):1985-94; Chapuis et al, Nat Med. 2019;25(7):1064-72). Methods:We report a phase I/II safety and dose escalation study evaluating WT1-TCR gene-modified autologous T cells in HLA-A*0201 positive patients with AML, MDS and CML, unsuitable for alloSCT (NCT02550535) (Fig 1A). Patient T cells were harvested by leucapheresis and transduced with a retroviral vector construct encoding the codon optimised variable and constant a and bchains of the human pWT126-specific TCR separated by a self-cleaving 2A sequence (Fig 1B). Bulk transduced T cells were analysed by flow cytometry (CD3, CD8 and Vb2.1) prior to infusion and at regular intervals post-infusion. A quantitative PCR assay was developed to identify WT1-TCR expressing T cells in the peripheral blood post infusion. Patients received minimal conditioning with fludarabine and methylprednisolone prior to transfer of transduced T cells. All subjects were followed for a minimum of 12 months or until death. Results:A total of 10 patients (6 AML, 3 MDS and 1 TKI- resistant CML) were recruited. The mean age was 71.3 years (range 64-75) and all had high risk disease (by cytogenetic or clinical criteria). All AML patients were in complete morphological remission at the time of trial entry, whilst MDS patients had ≤ 15% blasts on bone marrow examination. All 10 patients received the gene-modified T cells in dose escalation cohorts (seven patients received £2x107/kg and three patients received £1x108/kg bulk WT1 TCR transduced cells). No adverse events directly attributable to the investigational product were recorded apart from one possible cytokine release syndrome, which was managed without tociluzimab. Transferred T cells demonstrated in vivoproliferation commensurate with maintenance of functional capacity despite ex vivo manipulation (Fig 1C and 1D). The TCR-transduced T cells were detectable in all patients at 28 days and in 7 patients persisted throughout the study period (Fig 1E). All 6 AML patients were alive at last follow up (median 12 months; range 7-12.8 months). The 3 patients with MDS had a median survival of 3 months (range 2.1-3.96 months) post T cell infusion. 2 died from progressive disease and one from other causes. 2 patients discontinued the study early due to disease progression. Conclusions: This is the second reported phase I/II clinical trial of autologous WT1-TCR gene-modified T cells for treatment of AML and MDS in a high-risk cohort of patients not suitable for alloSCT. We have shown that the WT1-TCR T cells demonstrated a strong safety profile without detectable on-target, off-tumour toxicity and no severe adverse events in the ten patients treated. An important cause of treatment failure for adoptive cellular therapies is the lack of persistence of transferred T cells leading to loss of disease specific effects. We demonstrated that autologous WT1-TCR T cells proliferated in vivoand persisted for many months. Recent work within our group (in press) has shown that TCRs modified to include key framework residues, show increased TCR expression and functional improvement. These modifications could be incorporated into future studies to improve efficacy. This data supports the rationale for a larger, phase II trial of WT1-TCR T cells in myeloid malignancies in patients for whom alloSCT is not appropriate, in order to assess clinical efficacy. Figure 1 Disclosures Morris: Quell Therapeutics: Consultancy, Other: Scientific Founder,stock; Orchard Therapeutics: Consultancy. Qasim:CellMedica: Research Funding; Bellicum: Research Funding; UCLB: Other: revenue share eligibility; Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; Servier: Research Funding. Mount:Gamma Delta Therapeutics: Employment. Inman:Cellmedica: Employment. Gunter:Cellmedica: Employment. Stauss:Cell Medica: Other: I have stock; Quell Therapeutics: Consultancy, Other: I have stock.

pretreatment baseline to 3 months after starting vitamin D therapy. RESULTSOf the 26 patients, five (20%) responded to vitamin D; the mean (range) reduction in PSA level was 45.3 (15.9-95

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The persistence of antigen and chronic stimulation of T cells results in T cells’ functional decay, referred to as T cell dysfunction or exhaustion (T EX). T EXis an essential part of the checks and balances of the immune system chronic T cell stimulation. Assays that model T cell dysfunction/exhaustion with functional readouts are urgently needed for identification and characterization of active drug candidates. Here, to recapitulate persistent of antigen as it happens in tumor microenvironment, we have optimized an in vitrostimulation set-up using PBMCs from various donors that models key features of T cell exhaustion/dysfunction including main markers and functional features of T cell exhaustion. Various stimulation conditions were assessed, with stimulation lasting up to four rounds, and optimal conditions were selected for anti-CD3/anti-CD28 stimulations of PBMCs that resulted in a) increased expression of the co-inhibitory molecules associated with exhausted T cells, PD-1, TIM3, LAG3, CD38, and CD39, and b) impaired T cell functions as demonstrated by reduced expression of IFN-γ, TNF-α, and reduced cytotoxic killing in an allogeneic setting, using both a 2D cancer cell line and a 3D tumoroid model. This in vitroT cell dysfunction/exhaustion platform is a candidate to test drug candidates for their ability to delay or partially reverse T cell exhaustion. Crown Bioscience internal R&D