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Targeting tumor collagen hits the immunotherapeutic sweet spot

May 8, 2019

While immune checkpoint inhibitors and cytokine treatments have demonstrated efficacy against several types of cancer, they frequently cause severe side effects due to excessive and widespread off-tumor activation of the immune system. In a paper recently published in Science Translational Medicine, Ishihara and Ishihara et al. aimed to decrease off-tumor toxicities by taking advantage of the leaky tumor vasculature and targeting systemically administered immunotherapy to the collagen in the tumor stroma.

For a collagen-binding domain (CBD), the researchers chose the A3 domain of the von Willebrand factor, which binds to the collagen under the endothelial cells of a damaged blood vessel and initiates blood clotting. The CBD was attached to anti-CTLA-4 and anti-PD-L1 via chemical conjugation and to IL-2 via recombinant fusion. All three molecules bound collagen with sub-micromolar affinity, while fully retaining recognition of their targets. When the CBD protein alone was intravenously injected into mice bearing orthotopic MMTV-PyMT breast cancer, it preferentially localized to the tumor, and to a lesser extent, to the liver and kidneys. Similarly, CBD-anti-PD-L1, but not unmodified anti-PD-L1, localized to the tumor stroma. In addition, CBD-IL-2 accumulated around blood vessels in cryopreserved human melanoma tissue sections, while unmodified IL-2 did not.

In B16F10 tumor-bearing mice, the concentrations of CBD-anti-PD-L1 and CBD-anti-CTLA-4 in the blood were lower than their unmodified counterparts following systemic injection, likely due to their sequestration within the tumor. Combination of unmodified anti-CTLA-4 and anti-PD-L1 (CPI: checkpoint inhibitors) increased TNFα in the serum (indicating increased systemic activation of the immune system) and induced leukocyte infiltration into the lungs, CD8+ T cell infiltration into the liver, and morphological and biochemical changes to the liver (indicating liver damage). In contrast, combination of CBD-anti-CTLA-4 and CBD-anti-PD-L1 (CBD-CPI) led to reduced lung and liver toxicities compared to unmodified CPI and did not significantly raise serum TNFα levels. While unmodified IL-2 increased the spleen size and induced pulmonary edema in tumor-bearing mice, CBD-IL-2 did not. Together, these experiments demonstrate that targeting the tumor via collagen binding sequesters immunotherapeutic drugs within the tumor and reduces their systemic toxicity.

Ishihara and Ishihara et al. then examined the antitumor efficacy of the CBD-modified immunotherapies in three different tumor models. In mice with B16F10 melanoma, unmodified CPI slightly reduced tumor growth, while CBD-CPI increased this antitumor effect. Interestingly, systemic administration of the CBD-modified combination treatment showed similar efficacy to peritumoral administration of anti-CTLA-4 and anti-PD-L1 that had been conjugated with an extracellular matrix-binding peptide. A similar increase in antitumor efficacy with CBD-CPI was observed in CT26 colon carcinoma and MMTV-PyMT breast cancer models. In mice with breast cancer, CBD-CPI prolonged survival, resulted in complete remission in 6 of 12 mice, and protected survivors from rechallenge, indicating the establishment of immunological memory. Combination of CBD-CPI and CBD-IL-2 significantly suppressed the growth of MMTV-PyMT tumors and induced complete remission in 9 of 13 mice, whereas combination of unmodified CPI and IL-2 resulted in complete remission in only 1 of 13 mice. CBD-IL-2 alone delayed tumor growth in all three tumor models, while unmodified IL-2 showed no antitumor effect at the equivalent dose. CBD-IL-2 also slightly delayed the growth of the immune-excluded EMT6 breast tumors, which did not respond to unmodified IL-2 nor to CBD-CPI.

Seeking to uncover the mechanism behind the superior antitumor efficacy of CBD-modified immunotherapies, the researchers analyzed the T cell response in B16F10 tumor-bearing mice. CBD-CPI increased the frequency of CD8+ T cells and decreased Tregs within the tumor, while unmodified CPI did not. CD8+ T cells extracted from the tumor and stimulated ex vivo showed increased production of effector cytokines (IL-2, TNFα, IFNγ) after CBD-CPI, but not after unmodified CPI treatment. Both CBD-CPI and unmodified CPI treatments reduced intratumoral macrophages, MDSCs, and DCs. Similarly, CBD-IL-2 increased the number and frequency of CD8+ T cells in B16F10 and MMTV-PyMT tumors.

Thus, Ishihara and Ishihara et al. demonstrated that systemically-administered, CBD-modified immunotherapies localized to the tumor and increased the frequency and activation state of intratumoral CD8+ T cells, leading to improved antitumor response and reduced risk of toxicities in multiple tumor models. The results of this study, together with the simplicity of the CBD-conjugation approach, hold promise for clinical translation.

by Anna Scherer

Meet the researcher

This week, we interviewed Jun Ishihara, first co-author with Ako Ishihara.

What prompted you to tackle this research question?
Cancer immunotherapies using interleukin-2 (IL-2) or checkpoint inhibitor antibodies (CPI Ab) are FDA-approved drugs. They exhibit considerable antitumor activity in the clinic, but only a portion of patients benefited from these therapies. Also, a substantial number of patients suffered from treatment-related adverse events. In patients receiving combination CPI therapy, 96% experienced adverse events and 36% could not continue with the therapy due to adverse events. Immunotherapies serve to activate immune responses, and as such, side effects include the symptoms of systemic lymphocyte activation and autoimmune disease induction, which typically results from drug action in healthy organs. Patients experiencing adverse events have indeed ‘responded’ to the therapy in an undesired way, as their immune systems have been re-activated by treatment. Thus, we are seeking a way to increase antitumor response and reduce toxicity by developing a technology to target the tumor.

What was the most surprising finding of this study for you?
We were surprised by its improved efficacy. When we combined tumor-targeted checkpoint inhibitor and IL-2, most of the breast cancer-bearing mice were cured.

What was the coolest thing you’ve learned (about) recently outside of the lab?
We have recently learned that engineering approaches for cancer immunotherapy are getting more attention from researchers, doctors, and pharmaceutical companies. We believe that immuno-engineering will solve issues in the clinic, and contribute to the cancer patients in the near future.


Ishihara J., Ishihara A., Sasaki K., Lee S.S., Williford J.M., Yasui M., Abe H., Potin L., Hosseinchi P., Fukunaga K., Raczy M.M., Gray L.T., Mansurov A., Katsumata K., Fukayama M., Kron S.J., Swartz M.A., Hubbell J.A. Targeted antibody and cytokine cancer immunotherapies through collagen affinity. Sci Transl Med. 2019 Apr 10.

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