Article Text
PDF
Abstract
Background Interleukin 12 (IL-12) is a potent proinflammatory cytokine known for its ability to modulate the tumor microenvironment, enhancing adoptive cell therapies (ACT) in various preclinical models. However, its clinical utility has been limited by severe systemic toxicity.
Methods We developed IL-12TM-D, a drug-regulatable, inducible, and membrane-bound IL-12 construct for ACT in advanced cancers. IL-12TM-D integrates a lenalidomide-modulatory degron to exploit lenalidomide’s binding to Cereblon, an E3-ubiquitin ligase that degrades target substrates via proteasome-mediated degradation (figure 1A). Further control of IL-12 expression was achieved by placing IL-12TM-D downstream of an NFAT promoter, linking IL-12 expression to T-cell activation without the need for continuous lenalidomide administration. Additionally, the B7-1 transmembrane domain was introduced to minimize IL-12’s systemic release.
Results We demonstrated time and dose-dependent downregulation of IL-12TM-D expression in response to lenalidomide treatment (t1/2 of 3.98 h and IC50 of 18.39 nM). When combined with a model TCR targeting the p53R175H neoantigen (p53 TCR), IL-12TM-D expression was induced by TCR activation through co-culture with human ovarian cancer TYK-nu cells that expressed p53R175H (figure 1B). Functionally, IL-12TM-D enhanced the anti-tumor efficacy of T cells in various in vitro models, including human p53 or RAS neoantigen-targeting TCRs, human melanoma TILs targeting autologous tumor cells, and CD19 chimeric antigen receptor (CAR) cells against CD19-low leukemia cells. Notably, while CD19 CAR and IL-12TM-D co-expression did not show synergistic killing of CD19-high NALM6 human leukemia cells, it led to enhanced killing of CD19-low NALM6 cells. Mechanistically, CD4+ cells exhibited increased cytotoxicity when the p53 TCR was co-expressed with IL-12TM-D in CD4+ T cells, indicating IL-12 expression in CD4+ T cells contributes to enhanced anti-tumor efficacy of ACT. In a mouse xenograft model, IL-12TM-D expression in T cells engineered with the p53 TCR led to significant regression of TYK-nu ovarian cancer cells and improved survival (figure 2). In fact, co-expression of the p53 TCR and IL-12TM-D achieved complete tumor regression and long-term survival of all the 5 mice that were treated, which the p53 TCR alone failed to achieve (N=5, p<0.01; figure 2).
Conclusions We demonstrate IL-12TM-D, while allowing tight regulation of its expression, can enhance anti-tumor efficacy of ACT. These data support clinical evaluation of IL-12TM-D in the setting of ACT against advanced cancers. Lenalidomide has been approved for the treatment of multiple myeloma and myelodysplastic syndromes, and its proven safety may facilitate the translation of IL-12TM-D into the clinic.
Design of drug-regulatable, inducible, and membrane-bound IL-12 (IL-12TM-D) (A) Schematic of single-chain IL-12 conjugated with the lenalidomide degron and its mode of degradation. CRBN, Cereblon. (B) Schematic of IL-12TM-D induction
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.