From “Cold” to “Hot” Tumors with Masked Interleukin-12

Immunotherapies such as CAR-T cells and checkpoint-blocking antibodies have revolutionized cancer treatment by inducing long-term responses in patients with hematological malignancies (e.g., lymphoma and acute lymphocytic leukemia) and solid cancers such as metastatic melanoma, respectively (Haslauer et al. 2021, Robert, C. 2020). The first monoclonal antibody drug targeting the cytotoxic T lymphocyte antigen 4 (CTLA-4) immune checkpoint (i.e., ipilimumab) was developed over a decade ago. It was soon followed by antibodies against Programmed cell death 1 (PD-1) receptor and ligand (PD-L1) (e.g., pembrolizumab and atezolizumab) (Robert, C. 2020).

By blocking such immunosuppressive targets, these monoclonal antibody drugs prevent cancer and tumor-infiltrating cells from inhibiting T cell responses. Therefore, immune checkpoint inhibitors are said to “release the breaks of the immune system,” promoting T cell infiltration and cytotoxic tumor cell killing. Unfortunately, despite their great efficacy, the solid tumor “cold” landscape continues to present significant challenges to immunotherapy’s success in most patients.

Immune checkpoint inhibitors: Several monoclonal antibody drugs have been developed to target T cell inhibitory receptors or ligands expressed by cancer cells. “Immune checkpoint inhibitors approved by FDA. Pembrolizumab, Nivolumab, and Cemiplimab as anti-PD-1 antibodies, Ipilimumab as an anti-CTLA-4 antibody, as well as Atezolizumab, Avelumab, and Durvalumab as anti-PD-L1 antibodies.” Retrieved from Figure 1, Shiravand et al. 2022 without modifications.

Cold Tumor vs. Hot Tumor: What’s the Difference

The concepts of “Hot” and “Cold” tumors developed from the realization that different components within a tumor, beyond constituent cancer cells, define its immune status. It has been recognized that various elements within the tumor microenvironment, as determined by the extracellular matrix, blood supply, stromal cells, infiltrating cells, and soluble factors, critically shape the overall response to therapy.

Hot tumors are characterized by the presence of pro-inflammatory factors (e.g., interferons) that promote cytotoxic T cell infiltration and thus anti-tumor responses. In contrast, in cold tumors, immunosuppressive components such as anti-inflammatory cytokines (e.g., IL-6 and IL-10), T reg cells, and pro-tumorigenic macrophages promote immune escape. Immunotherapies, such as checkpoint inhibitors, fail against the anti-inflammatory conditions within cold tumors, which prevent sufficient cytotoxic T cell infiltration (Hernandez et al. 2021).

Cold and Hot tumors. “Main cells and signaling components involved in tumor microenvironment (TME) that confers lack (“cold” tumors) or enhancement (“hot” tumors) of immunogenicity to cancer cells.” Retrieved without modification from Figure 2, Hernandez et al. 2021.

Targeting Cold Tumors with Engineered Interleukin-12

Because the response to checkpoint inhibitors may be improved by promoting a hot tumor microenvironment, scientists at the Pritzker School of Molecular Engineering, University of Chicago, led by Dr. Jeffrey Hubbell, have developed a new approach to harness the pro-inflammatory properties of Interleukin-12 (Mansurov et al. 2022). Working on the hypothesis that targeting tumors with Interleukin-12 could induce the release of anti-tumorigenic factors (i.e., Interferon-gamma or IFN-gamma) and drive the recruitment of immune cells, the team engineered a masked form of Interleukin-12.

Based on their previous preclinical findings, Interleukin-12 accumulation within tumors, following systemic administration, could drive sufficient inflammation to activate innate and adaptive immune responses, inducing tumor regression in mice with cold tumors (Mansurov et al. 2020). Nevertheless, clinical findings have also identified adverse events associated with Interleukin-12, such as cytokine release syndrome and liver damage. Thus the team went on to engineer a non-signaling form of Interleukin-12 that could become preferentially activated within the tumor milieu.

For their experiments, Mansurov and colleagues relied on GenScript’s services to synthesize sequences to both mouse and human Interleukin-12 (i.e., p35 and p40 subunits) and their cloning into a mammalian expression vector, pcDNA3.1(+). To generate an inactivated form of the Interleukin-12 protein, the team engineered a modified sequence, connecting the N terminus of the p35 subunit to a mask, Interleukin-12Rβ1Q20–A261, via a linker sensitive to protease cleavage.

Masked Interleukin-12 cytokine. Recombinant Interleukin-12 was engineered by Mansurov et al. 2022 to be expressed as an inactive or non-signaling protein through masking with the Interleukin-12 receptor β1 extracellular domains. To support interaction with the p40 subunit, the Interleukin-12 receptor β1 mask was tethered to the p35 subunit through a linker containing consensus recognition sequences for tumor proteases.

“To produce recombinant human masked IL-12, a sequence encoding the human mask (derived from human IL-12Rβ1C24–E234) was fused to the N terminus of human p35 via a (G3S)11 linker and was subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript.” Mansurov et al. 2022

By following this approach, Mansurov and colleagues aimed to ensure that their newly engineered Interleukin-12 protein remained inactive in peripheral tissues while becoming activated within the tumor microenvironment where specific proteases abound. Among different masked Interleukin-12 variants developed, the team found that those having a linker with combined susceptibility to metalloproteinase and serine protease cleavage were more efficacious in vivo. Specifically, in a B16F10 mouse melanoma model, the variant M-L6-Interleukin-12 successfully induced strong anti-tumor activity, which was consistent with the presence of unmasked Interleukin-12 in recovered tumor tissue.

In a mouse model of subcutaneous MC38 colon adenocarcinoma, the masked Interleukin-12 variant was as efficacious as unmodified Interleukin-12 protein. Additionally, in mice bearing orthotopic EMT6 triple-negative breast tumors, M-L6-Interleukin-12 extended survival while a checkpoint inhibitor (i.e., anti-PD1) was ineffective. Significantly, the team noted that compared with unmodified Interleukin-12, systemic administration of M-L6-Interleukin-12 reduced adverse effects, as evidenced by unchanged body weight and lower levels of markers linked to potential liver and pancreas damage.

Lastly, by directly evaluating the tumors’ cellular and cytokine/chemokine profiles, Mansurov et al. confirmed that M-L6-Interleukin-12 led to the upregulation of various inflammatory factors mediating anti-tumorigenic responses and induced effective recruitment of antigen-presenting cells and cytotoxic T cells. Overall their findings demonstrated that M-L6-Interleukin-12 can re-shape the tumor microenvironment from a cold to a hot inflammatory state while having a more favorable safety profile than unmodified Interleukin-12. Therefore, these findings may open new opportunities for using engineered forms of Interleukin-12 in clinical studies.

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