Avidity optimization of a MAGE-A1-specific TCR with somatic hypermutation.

A T-cell receptor (TCR) with optimal avidity to a tumor antigen can be used to redirect T cells to eradicate cancer cells via adoptive cell transfer. Cancer testis antigens (CTAs) are attractive targets because they are expressed in the testis, which is immune-privileged, and in the tumor. However, CTAs are self-antigens and natural TCRs to CTAs have low affinity/avidity due to central tolerance. We previously described a method of directed evolution of TCR avidity using somatic hypermutation. In this study, we made several improvements to this method and enhanced the avidity of the hT27 TCR, which is specific for the cancer testis antigen HLA-A2-MAGE-A1278-286 . We identified eight point mutations with varying degrees of improved avidity. Human T cells transduced with TCRs containing these mutations displayed enhanced tetramer binding, IFN-γ and IL2 production, and cytotoxicity. Most of the mutations have retained specificity, except for one mutant with extremely high avidity. We demonstrate that somatic hypermutation is capable of optimizing avidity of clinically relevant TCRs for immunotherapy.

Optimizing T-cell receptor avidity with somatic hypermutation.

Adoptive transfer of T cells that have been genetically modified to express an antitumor T-cell receptor (TCR) is a potent immunotherapy, but only if TCR avidity is sufficiently high. Endogenous TCRs specific to shared (self) tumor-associated antigens (TAAs) have low affinity due to central tolerance. Therefore, for effective therapy, anti-TAA TCRs with higher and optimal avidity must be generated. Here, we describe a new in vitro system for directed evolution of TCR avidity using somatic hypermutation (SHM), a mechanism used in nature by B cells for antibody optimization. We identified 44 point mutations to the Pmel-1 TCR, specific for the H-2Db -gp10025-33 melanoma antigen. Primary T cells transduced with TCRs containing two or three of these mutations had enhanced activity in vitro. Furthermore, the triple-mutant TCR improved in vivo therapy of tumor-bearing mice, which exhibited improved survival, smaller tumors and delayed or no relapse. TCR avidity maturation by SHM may be an effective strategy to improve cancer immunotherapy.

Disruption of SUV39H1-Mediated H3K9 Methylation Sustains CAR T-cell Function.

Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.

DSP107 combines inhibition of CD47/SIRPα axis with activation of 4-1BB to trigger anticancer immunity.

BACKGROUND: Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPα and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPα 'don't eat me' signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. METHODS: Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. RESULTS: Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPα-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. CONCLUSIONS: DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients.

Toxicology and Pharmacokinetic Studies in Mice and Nonhuman Primates of the Nontoxic, Efficient, Targeted Hexameric FasL: CTLA4-FasL.

Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.

Regulation of SIRT1 protein levels by nutrient availability.

The mammalian NAD+ dependent deacetylase, SIRT1, was shown to be a key protein in regulating glucose homeostasis, and was implicated in the response to calorie restriction. We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions. These results have important implications on the relationship between sirtuins, nutrient availability and aging.

Regulation of SIRT6 protein levels by nutrient availability.

Sirtuins have been shown to regulate life-span in response to nutritional availability. We show here that levels of the mammalian sirtuin, SIRT6, increased upon nutrient deprivation in cultured cells, in mice after fasting, and in rats fed a calorie-restricted diet. The increase in SIRT6 levels is due to stabilization of SIRT6 protein, and not via an increase in SIRT6 transcription. In addition, p53 positively regulates SIRT6 protein levels under standard growth conditions but has no role in the nutrient-dependent regulation of SIRT6. These observations imply that at least two sirtuins are involved in regulation of life-span by nutrient availability.