De novo induction of intratumoral lymphoid structures and vessel normalization enhances immunotherapy in resistant tumors.

The tumor microenvironment confers profound resistance to anti-cancer immunotherapy. By targeting LIGHT, a member of the TNF superfamily of cytokines, to tumor vessels via a vascular targeting peptide (VTP), we developed a reagent with the dual ability to modulate the angiogenic vasculature and to induce tertiary lymphoid structures (TLSs). LIGHT-VTP triggered the influx of endogenous T cells into autochthonous or syngeneic tumors, which are resistant to immunotherapy. LIGHT-VTP in combination with checkpoint inhibition generated a large number of intratumoral effector and memory T cells with ensuing survival benefits, while the addition of anti-tumor vaccination achieved maximal therapeutic efficacy. Thus, the combination treatments stimulated the trafficking of pre-existing endogenous effector T cells as well as their intratumoral activation and were more successful than current immunotherapies, which fail due to tumor-intrinsic resistance mechanisms.

Immunomodulation of Tumor Vessels: It Takes Two to Tango.

The density of intratumoral CD8+ T cells predicts patient survival and responsiveness to immunotherapy. Effector T cell infiltration in turn is controlled by the tumor vasculature which co-evolves together with an immune-suppressive environment. At the T cell-vascular interface, endothelial cells actively suppress T cell trafficking and function. Conversely, forced activation, normalization, and differentiation of tumor vessels into high endothelial venule entrance portals for lymphocytes can facilitate T cell extravasation. Emerging evidence demonstrates that this process is not exclusively controlled by the endothelium. Indeed, tumor vasculature and CD4+ and/or CD8+ T cells may regulate each other: increasing local effector T cell numbers or re-invigorating pre-existing T cells via immune checkpoint blockade can directly affect the vasculature. A deeper understanding of the orchestration and duration of this reciprocal relationship may help shape the design of future immunotherapies.

Vascular targeting of LIGHT normalizes blood vessels in primary brain cancer and induces intratumoural high endothelial venules.

High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Immune priming and induction of tertiary lymphoid structures in a cord-blood humanized mouse model of gastrointestinal stromal tumor.

Gastrointestinal stromal tumors (GISTs) harbor diverse immune cell populations but so far immunotherapy in patients has been disappointing. Here, we established cord blood humanized mouse models of localized and disseminated GIST to explore the remodeling of the tumor environment for improved immunotherapy. Specifically, we assessed the ability of a cancer vascular targeting peptide (VTP) to bind to mouse and patient GIST angiogenic blood vessels and deliver the TNF superfamily member LIGHT (TNFS14) into tumors. LIGHT-VTP treatment of GIST in humanized mice improved vascular function and tumor oxygenation, which correlated with an overall increase in intratumoral human effector T cells. Concomitant with LIGHT-mediated vascular remodeling, we observed intratumoral high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), which resemble spontaneous TLS found in GIST patients. Thus, by overcoming the limitations of immunodeficient xenograft models, we demonstrate the therapeutic feasibility of vascular targeting and immune priming in human GIST. Since TLS positively correlate with patient prognosis and improved response to immune checkpoint inhibition, vascular LIGHT targeting in GIST is a highly translatable approach to improve immunotherapeutic outcomes.

Pericyte phenotype switching alleviates immunosuppression and sensitizes vascularized tumors to immunotherapy in preclinical models.

T cell-based immunotherapies are a promising therapeutic approach for multiple malignancies, but their efficacy is limited by tumor hypoxia arising from dysfunctional blood vessels. Here, we report that cell-intrinsic properties of a single vascular component, namely the pericyte, contribute to the control of tumor oxygenation, macrophage polarization, vessel inflammation, and T cell infiltration. Switching pericyte phenotype from a synthetic to a differentiated state reverses immune suppression and sensitizes tumors to adoptive T cell therapy, leading to regression of melanoma in mice. In melanoma patients, improved survival is correlated with enhanced pericyte maturity. Importantly, pericyte plasticity is regulated by signaling pathways converging on Rho kinase activity, with pericyte maturity being inducible by selective low-dose therapeutics that suppress pericyte MEK, AKT, or notch signaling. We also show that low-dose targeted anticancer therapy can durably change the tumor microenvironment without inducing adaptive resistance, creating a highly translatable pathway for redosing anticancer targeted therapies in combination with immunotherapy to improve outcome.

Therapeutic Induction of Tertiary Lymphoid Structures in Cancer Through Stromal Remodeling.

Improving the effectiveness of anti-cancer immunotherapy remains a major clinical challenge. Cytotoxic T cell infiltration is crucial for immune-mediated tumor rejection, however, the suppressive tumor microenvironment impedes their recruitment, activation, maturation and function. Nevertheless, solid tumors can harbor specialized lymph node vasculature and immune cell clusters that are organized into tertiary lymphoid structures (TLS). These TLS support naïve T cell infiltration and intratumoral priming. In many human cancers, their presence is a positive prognostic factor, and importantly, predictive for responsiveness to immune checkpoint blockade. Thus, therapeutic induction of TLS is an attractive concept to boost anti-cancer immunotherapy. However, our understanding of how cancer-associated TLS could be initiated is rudimentary. Exciting new reagents which induce TLS in preclinical cancer models provide mechanistic insights into the exquisite stromal orchestration of TLS formation, a process often associated with a more functional or "normalized" tumor vasculature and fueled by LIGHT/LTα/LTß, TNFα and CC/CXC chemokine signaling. These emerging insights provide innovative opportunities to induce and shape TLS in the tumor microenvironment to improve immunotherapies.

Remodeling of Metastatic Vasculature Reduces Lung Colonization and Sensitizes Overt Metastases to Immunotherapy.

Due to limited current therapies, metastases are the primary cause of mortality in cancer patients. Here, we employ a fusion compound of the cytokine LIGHT and a vascular targeting peptide (LIGHT-VTP) that homes to angiogenic blood vessels in primary tumors. We show in primary mouse lung cancer that normalization of tumor vasculature by LIGHT-VTP prevents cancer cell intravasation. Further, LIGHT-VTP efficiently targets pathological blood vessels in the pre-metastatic niche, reducing vascular hyper-permeability and extracellular matrix (ECM) deposition, thus blocking metastatic lung colonization. Moreover, we demonstrate that mouse and human metastatic melanoma deposits are targetable by VTP. In overt melanoma metastases, LIGHT-VTP normalizes intra-metastatic blood vessels and increases GrzB+ effector T cells. Successful treatment induces high endothelial venules (HEVs) and lymphocyte clusters, which sensitize refractory lung metastases to anti-PD-1 checkpoint inhibitors. These findings demonstrate an important application for LIGHT-VTP therapy in preventing metastatic development as well as exerting anti-tumor effects in established metastases.

Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell-Mediated Immunotherapy.

T-cell infiltration of solid tumors is associated with improved prognosis and favorable responses to immunotherapy. Mechanisms that enable tumor infiltration of CD8+ T cells have not been defined, nor have drugs that assist this process been discovered. Here we address these issues with a focus on VE-cadherin, a major endothelial cell-specific junctional protein that controls vascular integrity. A decrease in VE-cadherin expression is associated with tumor pathology. We developed an oligonucleotide-based inhibitor (CD5-2), which disrupted the interaction of VE-cadherin with its regulator miR-27a, resulting in increased VE-cadherin expression. Administration of CD5-2 in tumor-bearing mice enhanced expression of VE-cadherin in tumor endothelium, activating TIE-2 and tight junction pathways and normalizing vessel structure and function. CD5-2 administration also enhanced tumor-specific T-cell infiltration and spatially redistributed CD8+ T cells within the tumor parenchyma. Finally, CD5-2 treatment enhanced the efficacy of anti-PD-1 blocking antibody. Our work establishes a role for VE-cadherin in T-cell infiltration in tumors and offers a preclinical proof of concept for CD5-2 as a therapeutic modifier of cancer immunotherapy via effects on the tumor vasculature. Cancer Res; 77(16); 4434-47. ©2017 AACR.

Intratumoral LIGHT Restores Pericyte Contractile Properties and Vessel Integrity.

Normalization of the tumor vasculature is an emerging concept shown to improve anti-cancer therapy. However, there are currently no clinical interventions that effect long-lasting normalization. Here, we have developed a strategy for normalization by specific intratumoral delivery of LIGHT/TNFSF14. Importantly, normalization occurs by induced expression of contractile markers in intratumoral pericytes, which in turn re-establishes tight pericyte-vessel alignment. Restoring vessel integrity improves tumor perfusion and acts as adjuvant to chemo- and immunotherapy. Mechanistically, intratumoral LIGHT induces pericyte differentiation and normalization via Rho kinase signaling. Minute amounts of LIGHT act in a paracrine fashion to trigger an amplifying cascade involving transforming growth factor ß (TGF-ß) from peri-vascular macrophages. That these effects can be reproduced by adoptive transfer of LIGHT-stimulated macrophages alone demonstrates their central role in regulating pericyte differentiation. Our findings highlight a crucial role of pericyte contractile properties in vascular normalization, effected by macrophage signaling, thus providing so far unexplored anti-cancer opportunities.