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Abstract
Background Our goal is to create an effective and novel immunotherapy against multiple solid tumors with minimal toxicity. IL-12 is an ideal immune-activator, capable of activating both innate and acquired immune response.1 Peripheral toxicity limits the use of systemic IL-12 therapy for solid tumors in clinical practice. We have hypothesized that the tumor-specific drug delivery system would improve IL-12 immunotherapies and took original approaches to use protein-based engineering (figure 1).
Methods We developed technologies to turn the tumor collagen into a drug reservoir through both intravenous injections. The recombinant fusion of IL-12 to a CBD allows for targeted IL-12 delivery into the highly collagenous tumor stroma.1 2 Here, we evaluated the pharmacology, safety, efficacy and the effects of CBD-IL-12 to the tumor microenvironment. We used multiple metastasis models, recurrence models, humanized mice, and dogs.
Results Intravenously-injected CBD proteins preferentially localized to the tumor, but not in other organs in mice (figure 2). CBD-IL-12 (recombinant fusion protein) eradicated immunologically cold and checkpoint inhibitor-unresponsive breast tumors (figure 2B). CBD-IL-12-treated mice that were tumor-free developed systemic immunological memory, as 12 mice out of 13 rejected rechallenge with the breast tumor cells in their contralateral mammary fat pad (figure 2B). Single injection of CBD-IL-12 induced 67% complete remission of B16F10 tumors. CBD fusion to IL-12 reduced the systemic toxicity, such as hepatotoxicity markers and cytokine storm (figure 2C,D). CBD-IL-12 treatment increased intratumoral cytokine and immune cell numbers (figure 2E-H). In short, this tumor collagen-targeting approach improves both safety and efficacy of IL-12. We showed that the CBD can target multiple solid tumor types, because the CBD exploits the tumor vasculature-specific pathological structure (e.g. leakiness and exposure of collagen to the bloodstream). Recently, we have shown that CBD-IL-12 has significant anti-tumor efficacy in breast, lung, ovarian, colon, prostate, melanoma, head and neck, pancreatic cancers and melanoma, sarcoma, and glioblastoma in syngeneic mouse models.1–7 CBD-IL-12 is effective in cold tumors, low-mutation burden tumors , and prevents metastasis and recurrence. CBD-IL-12 stimulated antigen-presenting cells and T cells.Also, humanized mice with humanized CBD-IL-12 show great anti-tumor effects in patient-derived tumors. Humanized CBD-IL-12 was tested in dogs, and showed great tolerability at the therapeutic dose. Human CBD-IL-12 molecule was successfully manufactured in a clinical-grade.
Conclusions CBD-IL-12 is effective to multiple difficult, immunologically cold tumors with low mutation burdens. Collagen is an attractive target because abnormally high expression of collagen is a shared nature of tumors, regardless of tumor types and patients.
Acknowledgements I thank collaborators. Fundings: Prostate cancer UK, Sarcoma UK, Brain tumour charity, University of Chicago, and Cancer research UK
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Scheme of CBD-IL-12 action. Possible immunological actions after CBD-IL-12 production. Locally produced CBD-IL-12 is retained in the tumour due to collage affinity, whereas wt IL-12 diffuses to the systemic body. We expect that tumour retention of CBD-IL-12 leads to enhanced efficacy and safety. CBD-IL-12 binds to T/NK cells and then we expect three effects. 1) CBD-IL-12 will activate T cells for enhanced tumour cell killing. 2) IFNγ will be produced within the TME, activating antigen-presenting cells, priming immune effectors to instill immunological memory upon subsequent challenges. 3) IFNγ within the TME will increase the intratumoral chemokine concentration, recruiting immune cells.
CBD-IL-12 protein therapy enhances both efficacy and safety compared to wild-type IL-12 in solid tumour models. (A) Intravenously-injected fluorescently-tagged CBD protein preferentially accumulates in the tumour (B) CBD fusion to IL-12 extends survival of EMT6 breast tumour-bearing mice (immunologically cold). IL-12, or equimolar CBD-IL-12 was injected once. 13 CBD-IL-12 treated mice out of 15 showed complete response (CR). 12 tumour-free mice out of 13 rejected rechallenge with EMT6 cells in their contralateral mammary fat pad. (C-D) CBD-fusion to IL-12 reduced day 2 liver damage marker (ALT) activity and IFNγ levels in the serum. (E-F) CBD-fusion to IL-12 increased intratumoral IFNγ and CXCL10 levels and (G-H) frequency of Cd8+ T cells and CD103+ migratory dendritic cells.
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