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1213 Tumor-ECM targeted IL-12: improved safety and efficacy in clinically relevant settings
  1. Jun Ishihara
  1. Imperial College London, London, UK
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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


  1. Mansurov A, Ishihara J, Hosseinchi P, Ishihara A, Marchell MT, Williford JM, Potin L, Alpar AT, Raczy MM, Swartz MA, Hubbell JA. Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours. Nature Biomedical Engineering, April 2020 10.1038/s41551–020-0549–2, 2020. PMID:32284554

  2. Ishihara J, Ishihara A, Sasaki K, Lee S, Williford JM, Yasui M, Abe H, Potin L, Hosseinchi P, Fukunaga K, Grey LT, Kron S, Swartz MA, Hubbell JA. Targeted antibody and cytokine cancer immunotherapies through collagen affinity. Science Translational Medicine, 2019;11:eaau3259. PMID: 30971453 PMCID: PMC6541444

  3. Williford JM, Ishihara J, Ishihara A, Mansurov A, Hosseinchi P, Marchell MT, Swartz MA, Hubbell JA. Recruitment of CD103+ DCs via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor. Science Advances, 2019;5:aay1357, PMID: 31844672 PMCID: PMC6905870

  4. Katsumata K, Ishihara J, Mansurov A, Ishihara A, Raczy MM, Yuba E, Hubbell JA. Targeting inflammatory sites through collagen affinity enhances the therapeutic efficacy of anti-inflammatory antibodies. Science Advances, 2019;5:aay1917, PMID: 31723606 PMCID: PMC6834392

  5. Bhatia V, Kamat NV, Pariva TE, Wu L-T, Tsao A, Sasaki K, Sun H, Javier G, Nutt S, Coleman I, Hitchcock L, Zhang A, Rudoy D, Gulati R, Patel RA, Roudier MP, True LD, Srivastava S, Morrissey CM, Haffner MC, Nelson PS, Priceman SJ, Ishihara J, Lee J. K Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell therapy. Narure Communications 2023;14:2041.

  6. Mansurov A, Hosseinchi P, Lauterbach A, Gray LT, Alpar AT, Budina E, Cao S, Solanki A, Gomes S, Williford JM, Swartz MA, Mendoza JL, Ishihara J, Hubbell JA. Eliminating the Immunotoxicity of Interleukin-12 through Protease-Sensitive Masking. Nature Biomedical Engineering, May 2022

  7. Sasaki K, Ishihara J, Ishihara A, Miura R, Mansurov A, Fukunaga K, Hubbell J.A. Engineered collagen-binding serum albumin as a drug-conjugate carrier for cancer therapy. Science Advances, 2019;5:eaaw6081. PMID: 31453327 PMCID: PMC6693903

Abstract 1213 Figure 1

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.

Abstract 1213 Figure 2

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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