Weekly Digests
‹ Back to May

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.

In the Spotlight...

Cancer stemness, intratumoral heterogeneity, and immune response across cancers

Utilizing an immune- and proliferation-depleted gene set that defined “stemness”, and data from TCGA, Miranda and Hamilton et al. performed an integrated analysis of 8,290 tumors across 21 solid tumor types. Tumor stemness varied across cancer types and was predictive of poor patient survival. Stemness negatively correlated with immune infiltration, type I IFN signaling, and endogenous retrovirus expression, and positively correlated with tumor heterogeneity, mutation load, expression of cancer testis antigen, and immunosuppression, supporting a hypothesis that stemness promotes immune evasion, resulting in cold TMEs that foster heterogeneity.

REVIEW: The clinical application of cancer immunotherapy based on naturally circulating dendritic cells

Dendritic cell vaccines using monocyte-derived DCs (moDCs) have shown low toxicity, but limited clinical efficacy, perhaps due to extensive ex vivo processing. Recent developments in magnetic bead technology have allowed for the quick isolation of naturally circulating DC (nDC) subsets (pDCs and cDC2s, with ongoing efforts to isolate CD141+ cDC1s), which may retain more functionality than moDCs. Thus far, nDCs have been used in 9 clinical trials, though available data is still limited. Additional ways of improving DC vaccines include optimizing the dosing and mode of administration, using neoantigens for loading, and designing rational combinations.

Restriction of PD-1 function by cis-PD-L1/CD80 interactions is required for optimal T cell responses

Sugiura et al. found that PD-L1 and CD80 bind in cis on primary dendritic cells, limiting the capacity of PD-L1 to interact with PD-1 on T cells, but not limiting the capacity of CD80 to interact with CD28 or CTLA-4. DC expression of mutated PD-L1 or CD80 incapable of cis binding significantly suppressed T cell responses in both murine and human co-cultures. In knock-in mice in which cis binding does not occur, antigen responses were reduced, antitumor activity following vaccination was suppressed, and autoimmune responses were minimized. DCs from the knock-in mice were less effective as vaccines than DCs from wild-type mice.

Fatty acid transport protein 2 reprograms neutrophils in cancer

Veglia et al. showed that in PMN-MDSCs (pathologically activated neutrophils) of tumor-bearing mice, the transcription factor STAT5 (likely activated by GM-CSF) upregulated the expression of FATP2, a fatty acid transport protein. FATP2 increased the uptake of arachidonic acid, a precursor for prostaglandin E2 (PGE2). Upregulated PGE2 in PMN-MDSCs mediated immune suppression and tumor growth. Similar results were observed in patients with head and neck, lung, and breast cancer. Selective targeting of PMN-MDSCs via inhibition of FATP2 decreased tumor growth, and combination with anti-CTLA-4 led to tumor rejection in most mice.

CLINICAL TRIAL: A safe and potent anti-CD19 CAR T cell therapy

Ying and Huang et al. screened variants of CD19-BBz (with 4-1BB and CD3ζ domains) CARs, and found that a variant (CD19-BBz(86)) with small insertions in the hinge/transmembrane domain reduced antigen-stimulated CAR T cell cytokine production and proliferation, but maintained cytolytic activity in vitro and in vivo. In a phase I trial, 25 patients with refractory B cell lymphoma were treated with CD19-BBz(86) CAR T cells; 15 patients responded, with 6/11 treated at the highest dose achieving CR. Cytokine levels remained at baseline, and none of the patients experienced severe cytokine release syndrome or neurological toxicity.

Targeting self and neo-epitopes with a modular self-adjuvanting cancer vaccine

Belnoue and Mayol et al. designed a DC-targeting chimeric protein vaccine consisting of a cell-penetrating peptide to enhance internalization, a multi-antigenic domain to introduce MHC-restricted peptides, and a TLR2/4 agonist domain to act as a self-adjuvant. The vaccine induced effector and memory CD4+ and CD8+ T cell responses to multiple epitopes, increased tumor infiltration, remodeled the TME, and enhanced tumor control in various murine models. Antibodies to the vaccine developed but did not impact T cell responses. A vaccine candidate for use in humans showed safety and immunogenicity in a non-human primate.

Everything New this Week In...