Background
Constructs that express cytokines in immune cells are a powerful potential add-on to cell therapies. Onboard, membrane-tethered cytokines can mitigate some of the shortcomings of exogenous cytokines, including systemic toxicity, short half-life, and poor tissue penetration. However, in the case of engineered logic gates, designed to improve tumor-specific killing by integrating signals from multiple antigens, the enhancements from onboard cytokines must not override the selectivity achieved by the logic-gate mechanism. For example, an ideal onboard booster would not interfere with the inhibitory receptor of a NOT gate, such as the LIR-1-based blocker used in the TmodTM dual-receptor system.1 Tmod is designed to exploit situations where healthy normal cells express the target of the blocker, while the tumor cells do not. Such situations occur, for example, when the cancer cells lack target expression due to loss of heterozygosity (figure 1),2 stem cell transplant, or epigenetic changes. The challenge is to demonstrate that onboard cytokines augment Tmod potency without sacrificing selectivity.
Methods
To identify cytokines that boost Tmod activity without overriding blocker function, systematic comparisons were made in acute and long-term in vitro tumor co-culture assays (figure 2) and in novel in vivo animal studies (figure 3), which include measurement of dose-response in mice with micrografts and in mice treated with surrogate Tmod constructs that recognize mouse homologs of the human target antigens.
Results
Large-effect cytokines (IL-2, IL-7, IL-12, IL-15, IL-18, IL-21) were tested in the context of the Tmod NOT gate. Membrane-tethered IL-12 boosted the antigen-dependent activity of Tmod constructs by ≥ 10-fold in a variety of longer-term in vitro and in vivo assays without overriding the blocker, a key requirement of booster function (figures 2 and 3). Minimal soluble IL-12 was observed in the blood of treated mice, consistent with an acceptable safety profile of a Tmod product that carries tethered IL-12. Membrane-tethered IL-12 also mitigated the immunosuppressive effect of TGFβ (figure 2C), thought to be a key component of the tumor microenvironment. Membrane-tethered IL-12 was studied not only in solid tumor Tmod constructs but also in Tmod constructs that target AML antigens.
Conclusions
The membrane-tethered IL-12 module can be combined with both blood and solid tumor Tmod constructs to boost efficacy while preserving selectivity.
References
DiAndreth B, et al. Clin Immunol. 2022;241:109030.
Hamburger AE, et al. Mol Immunol. 2020;128:298–310.
Abstract 341 Figure 1
Engineered T cells kill tumors but spare normal cells by leveraging loss of gene expression in tumors; eg, expression loss of 1 allele via loss of heterozygosity
Abstract 341 Figure 2
Mem-IL-12 boosts acute and long-term Tmod activity, while preserving selectivity. (A) Schematic of tethered IL-12 co-expressed with Tmod. (B) The specific killing of CEA Tmod cells constitutively expressing mem-IL-12 at 48 hrs with an E:T ratio of 1:9 to 27:1. Killing is enhanced with mem-IL-12, but selectively is preserved. Tumor: CEA(+) HLA-A*02(-) H508; ‘Normal’: CEA(+) HLA-A*02(+) H508. (C) Both consecutive-expressing (EF1a) and activation-induced (NFAT) mem-IL-12 enhance antigen-dependent expansion of MSLN Tmod upon repeated tumor challenges and partially overcome TGFB1 inhibition (10ng/ml TGFB1 added in co-culture) (y axis log10 scale)
Abstract 341 Figure 3
Mem-IL-12 boosts Tmod in vivo activity and expansion, while preserving selectivity. (A) Schematic of in vivo animal study. Tumor and ‘normal’ were established by engraftment of 5e4MSLN(+) HLA-A*02(-) MS751 cells (tumor; left flank) and 5e4 MSLN(+) HLA-A*02(+) MS751 cells (‘normal’; right flank), 7.5e4 T cells were infused 11 days later. (B) Tmod potency and selectivity were assessed via bioluminescence (BLI). C. Blood was collected to measure T cells expansion and IL-12 shedding