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Fighting toxicity with transience by using IL-12 mRNA

December 4, 2019

T cells that are genetically engineered to produce IL-12 are highly effective against tumors, but are also highly toxic. To overcome this toxicity, Etxeberria et al. engineered tumor-specific CD8+ T cells that transiently express IL-12 by electroporating them with mRNA. Their results, recently published in Cancer Cell, show that intratumoral adoptive transfer of these cells, alone or in combination with CD137 (i.e. 4-1BB) agonism, may be a highly effective strategy for clearing tumors.

To begin, Etxeberria et al. optimized a single chain (sc)IL-12 mRNA for expression following electroporation. The scIL-12 mRNA was then electroporated into OVA-specific OT-I TCR transgenic murine CD8+ T cells. Between 50 and 70 percent of cells remained viable after electroporation, and functional scIL-12 was expressed, eliciting IFNγ secretion and enhanced CD137 expression.

To test the efficacy of these cells for adoptive cell transfer, the researchers generated mice with bilateral B16OVA melanomas and injected the scIL-12-transduced OT-I cell product intratumorally into one side. This elicited antitumor responses in both injected and distant tumors, outperforming mock-transduced OT-I cells. The addition of agonistic anti-CD137, also delivered intratumorally, further enhanced the antitumor efficacy of scIL-12-transduced OT-I cells, most notably enhancing overall survival. Using OT-I mock-transduced T cells plus anti-CD137, or scIL-12 mRNA-transduced non-specific CD8+ T cells plus anti-CD137 showed limited efficacy in the same tumor model, indicating a requirement for tumor-specific T cells, IL-12 expression, and CD137 agonism for best results. Similar results were observed in mice bearing OVA-expressing Panc02 tumors, and in B16OVA-bearing mice modeling disseminated disease.

Given that OT-I cells have a high TCR affinity, the researchers tested whether T cells with a lower-affinity TCR would work as well. To test this, they transduced Pmel1 cells, which have an intermediate-affinity TCR specific for gp100 in B16 tumors, with scIL-12 mRNA and tested them in mice bearing bilateral B16OVA tumors. In combination with agonist anti-CD137, these cells effectively cleared injected tumors, though contralateral tumors eventually progressed. In this setting, the contribution of CD137 was more significant. The researchers later found that OT-I cells persisted longer in the treated tumor and trafficked to contralateral tumors more effectively than Pmel1 cells. These results may be explained by the weaker TCR affinity, or by weaker expression of gp100 compared to OVA in B16OVA tumors.

Looking more closely at the activity of scIL-12 mRNA-transduced OT-I T cells in tumors, the researchers found that levels of IL-12+ OT-I cells and IFNγ production by these cells peaked at 12 hours after electroporation, and remained detectable after 24 hours in both treated and untreated tumors. The researchers also observed an IFNγ-dependent increase in antigen presentation on cancer cells. Neutralization of IFNγ dampened the antitumor efficacy of scIL-12-producing OT-I cells, suggesting a dependence on IFNγ. Activation of myeloid-derived suppressor cells did not appear to play an important role in this setting, despite previous studies suggesting it might.

Using intravital microscopy, the researchers were able to track fluorescently tagged scIL-12 mRNA-transduced OT-I cells in B16OVA tumors implanted in the ear dermis. Compared to mock-transduced cells, these cells induced more apoptosis and traveled more rapidly within the tumor microenvironment, making more T cell-to-tumor contact interactions, but spending less time on each individual interaction. In bilateral tumor models, scIL-12 mRNA-transduced OT-I cells increased in frequency in tumors and tumor-draining lymph nodes on both sides compared to mock-transduced OT-I cells. Persistence was enhanced in scIL-12 mRNA-transduced OT-I cells, and CD137 agonism further enhanced persistence, as well as differentiation towards central memory and terminal effector T cell fates. Further, treatment with antigen-specific scIL-12-transduced T cells increased endogenous CD4+ and CD8+ T cell populations, reduced the number of Tregs, and reduced Treg proliferation.

Parsing out the role of endogenous immune cells, Etxeberria et al. tested their treatment regimen in Rag-/- mice, which lack B and T cells, and in Batf3-/- mice, which lack cDC1 dendritic cells. In both settings, bilateral tumors initially disappeared. While tumors did not return in wild-type mice, all Rag-/- mice and a large portion of Batf3-/- mice relapsed, suggesting that while adoptively transferred cells are sufficient to eliminate palpable tumors, endogenous immune cells are essential for durable responses. Investigating this further, the researchers observed that B16OVA-bearing mice treated with scIL-12 mRNA-transduced Pmel1 cells showed an increase in endogenous CD8+ T cells recognizing OVA, and that B16F10-bearing mice showed evidence of enhanced T cell recognition of the melanosomal antigen TRP2, suggesting that epitope spreading had occurred. This phenomenon was not observed in Batf3-/- mice, suggesting that epitope spreading is dependent on cDC1 dendritic cells.

In efforts to optimize their therapeutic regimen, Etxeberria et al. showed that preconditioning with single-dose total body irradiation and supportive treatment with IL-2 enhanced the efficacy of scIL-12 mRNA-transduced Pmel1 cell therapy and anti-CD137. The researchers also tested intravenous and subcutaneous delivery routes and found that they failed to stack up against intratumoral injection, as adoptively transferred cells arrived at the tumors later, missing the window for high scIL-12 mRNA expression in the tumor. To simplify the administration of agonistic anti-CD137, the researchers transduced OT-I cells with an mRNA encoding CD137L, which yielded functional CD137L expression. Co-transducing OT-I cells with scIL-12 mRNA and CD137L mRNA enhanced antitumor efficacy in a way that was comparable to agonist CD137 antibodies.

Overall, the researchers show that the combination of tumor antigen-specific T cells, transient IL-12 production, and CD137 agonism can effectively treat tumor-bearing mice. Exploring the possible clinical relevance of this strategy, the researchers transduced CD8+ T cells from TIL populations from murine MC38 and B16OVA tumors. Treatment with PD-1+, but not PD-1-, sorted cultures of these transduced cells cleared respective bilateral tumors. Further, TILs from human patients could be effectively transduced with human scIL-12 mRNA. In a case where patient-derived tumor could engraft into mice, IL-12 mRNA-transduced TILs from that patient controlled tumor progression, suggesting that clinical translation may be feasible and efficacious.

by Lauren Hitchings

Meet the Researcher

This week, lead author Ignacio Melero shares with us some insights about the study.

On the left, first author Iñaki Etxeberria and on the right, lead author Ignacio Melero.

What prompted you to tackle this research question?
We need to make adoptive T cell therapy work against solid tumors. Considering that the main limitations are T cell trafficking into the malignant tissue microenvironment and poor antigen presentation, we resolved to test intratumoral injection and engineered the cells so they would be geared to function in the hostile tumor microenvironment. To this end, we transiently modified gene expression with mRNAs encoding interleukin-12 and CD137-ligand. Of note, permanent transfection has been shown to create serious safety problems, especially with interleukin-12.

What was the most surprising finding of this study for you?
There were two most surprising findings: the first one is that the strategy works potently not only against the injected tumor, but against uninjected distant foci of disease. The second that the strategy promotes mechanisms that prime T cells against other antigens in the tumor and that these vaccinating phenomena are critical to prevent relapse.

What was the coolest thing you’ve learned (about) recently outside of work?
I have recently read a novel on double agents during the second world war by Ben Macyntire, teaching me what disinformation can achieve and I have recently swam for the first time in my life two kilometers non-stop.


Etxeberria I., Bolaños E., Quetglas J.I., Gros A., Villanueva A., Palomero J., Sánchez-Paulete A.R., Piulats J.M., Matias-Guiu X., Olivera I., Ochoa M.C., Labiano S., Garasa S., Rodriguez I., Vidal A., Mancheño U., Hervás-Stubbs S., Azpilikueta A., Otano I., Aznar M.A., Sanmamed M.F., Inogés S., Berraondo P., Teijeira Á., Melero I. Intratumor Adoptive Transfer of IL-12 mRNA Transiently Engineered Antitumor CD8(+) T Cells. Cancer Cell. 2019 Nov 6.

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To learn more, see this collection of figures at ReFigure.

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