Expression of a dominant T-cell receptor can reduce toxicity and enhance tumor protection of allogeneic T-cell therapy

Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of ‘dominant’ T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a ‘dominant’ T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in vitro selection of allogeneic T cells expressing high levels of a ‘dominant’ T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease

Safety and efficacy of Tet-regulated IL-12 expression in cancer-specific T cells

We explored whether engineering of T cell specificity and effector function improves immunotherapy of solid tumors. Although IL-12 can enhance cancer immunity, a strategy of safe IL-12 delivery without toxicity is currently lacking. We engineered T cells to express IL-12 controlled by the NFAT promoter responsive to TCR stimulation, or by the Tet-On promoter responsive to doxycycline. In vivo, NFAT-engineered T cells caused lethal toxicity, while Tet-engineered T cells were safe in the absence of doxycycline. Combining gene transfer of the melanoma-specific TRP2-TCR with Tet-IL-12 engineering revealed that temporal induction of IL-12 was essential to inhibit the growth of B16F10 melanoma tumors. Induced IL-12 increased the number of tumor-infiltrating T cells and also prevented the down-modulation of the TRP2-TCR and the associated up-regulation of the PD1 marker that was observed in the absence of IL-12. In addition, temporal induction of IL-12 expression also increased the number of plasmacytoid DC in the tumor micro-environment. We show that repeated induction of IL-12 can be used to enhance control of tumor growth without encountering systemic toxicity. The observation that TCR engineering combined with Tet-regulated IL-12 expression can achieve tumor immunity without the side effects that are usually associated with the in vivo use of IL-12 warrants translation of this concept into the clinic

The S enantiomer of 2-hydroxyglutarate increases central memory CD8 populations and improves CAR-T therapy outcome

Cancer immunotherapy is advancing rapidly and gene-modified T cells expressing chimeric antigen receptors (CARs) show particular promise. A challenge of CAR-T cell therapy is that the ex vivo-generated CAR-T cells become exhausted during expansion in culture, and do not persist when transferred back to patients. It has become clear that naive and memory CD8 T cells perform better than the total CD8 T-cell populations in CAR-T immunotherapy because of better expansion, antitumor activity, and persistence, which are necessary features for therapeutic success and prevention of disease relapse. However, memory CAR-T cells are rarely used in the clinic due to generation challenges. We previously reported that mouse CD8 T cells cultured with the S enantiomer of the immunometabolite 2-hydroxyglutarate (S-2HG) exhibit enhanced antitumor activity. Here, we show that clinical-grade human donor CAR-T cells can be generated from naive precursors after culture with S-2HG. S-2HG-treated CAR-T cells establish long-term memory cells in vivo and show superior antitumor responses when compared with CAR-T cells generated with standard clinical protocols. This study provides the basis for a phase 1 clinical trial evaluating the activity of S-2HG-treated CD19-CAR-T cells in patients with B-cell malignancies

Glutarate regulates T cell metabolism and anti-tumour immunity

Abstract T cell function and fate can be influenced by several metabolites: in some cases, acting through enzymatic inhibition of α-ketoglutarate-dependent dioxygenases, in others, through post-translational modification of lysines in important targets. We show here that glutarate, a product of amino acid catabolism, has the capacity to do both, and has potent effects on T cell function and differentiation. We found that glutarate exerts those effects both through α-ketoglutarate-dependent dioxygenase inhibition, and through direct regulation of T cell metabolism via glutarylation of the pyruvate dehydrogenase E2 subunit. Administration of diethyl glutarate, a cell-permeable form of glutarate, alters CD8+ T cell differentiation and increases cytotoxicity against target cells. In vivo administration of the compound is correlated with increased levels of both peripheral and intratumoural cytotoxic CD8+ T cells. These results demonstrate that glutarate is an important regulator of T cell metabolism and differentiation with a potential role in the improvement of T cell immunotherapy.Abstract T cell function and fate can be influenced by several metabolites: in some cases, acting through enzymatic inhibition of α-ketoglutarate-dependent dioxygenases, in others, through post-translational modification of lysines in important targets. We show here that glutarate, a product of amino acid catabolism, has the capacity to do both, and has potent effects on T cell function and differentiation. We found that glutarate exerts those effects both through α-ketoglutarate-dependent dioxygenase inhibition, and through direct regulation of T cell metabolism via glutarylation of the pyruvate dehydrogenase E2 subunit. Administration of diethyl glutarate, a cell-permeable form of glutarate, alters CD8+ T cell differentiation and increases cytotoxicity against target cells. In vivo administration of the compound is correlated with increased levels of both peripheral and intratumoural cytotoxic CD8+ T cells. These results demonstrate that glutarate is an important regulator of T cell metabolism and differentiation with a potential role in the improvement of T cell immunotherapy

Redirection to the bone marrow improves T cell persistence and antitumor functions

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15–dependent homeostatic expansion and promoted the differentiation of memory precursor–like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required

A Quick, Simple and Unbiased Method to Quantify C2c12 Myogenic Differentiation

Introduction: C2C12 myoblasts undergo in vitro myogenesis to form protein-rich multinucleated myotubes. Determining the fraction of total nuclei incorporated into myotubes is a commonly used method to quantify the extent of differentiation, but it is labor-intensive and susceptible to operator bias. Methods: We have developed a simple method to quantify myotube formation using micrographs of Jenner-Giemsastained C2C12 cultures. Because myotubes are darkly stained by Jenner-Giemsa dyes, the extent of myotube formation correlates with an increase in pixels attributed to the darkest tones. Thus, image histograms were obtained from photographs using ImageJ software, and the sum of the darkest tones was used as a measure of myotube density. Results: Measurements of myotube density mirrored those of fusion index during C2C12 differentiation and after treatment with prostaglandin D2, an inhibitor of C2C12 myogenesis. Conclusions: We propose this inexpensive, quick, and unbiased method to quantify C2C12 differentiation as a complement of the fusion index analysis. Muscle Nerve 44: 366-370, 201