Tumor-associated lymphatic vessels are clinically linked to aggressive tumor progression and metastasis. Their expansion is driven by vascular endothelial growth factor-C (VEGF-C), which is highly expressed in the tumor microenvironment (TME) in melanoma and other solid tumors. Although elevated VEGF-C promotes an immunosuppressive TME, it paradoxically also enhances the response to immunotherapy in mouse models and correlates with improved survival after checkpoint blockade in patients. This enhanced response was associated with VEGF-C-induced alterations in the tumor microenvironment before immunotherapy, characterized by increased infiltration of naïve T cells in VEGF-C-overexpressing tumors, which creates favorable conditions for local T cell activation. Here, we investigated how VEGF-C reshapes the tumor microenvironment during and after immunotherapy response to improve immunotherapy outcome in melanoma, focusing on immune cell trafficking and memory T cell development. We utilized a B16F10 melanoma model overexpressing VEGF-C (B16-VEGFC) or a control vector (B16-Control) in conjunction with the pmel adoptive cell transfer and peptide vaccination model. Our data demonstrate that B16-VEGFC tumors harbor increased transferred antigen-specific T cells, including more proliferating effector and central memory T cells, along with enhanced endogenous T and B cell populations. In parallel, B16-VEGFC tumors and their draining lymph nodes (tdLN) contain higher fractions of total memory T cells, and Tcf1+ stem-like memory T cells in lymphangiogenic tumors localize near lymphatic vessels. In addition to T cell memory, B16-VEGFC tumors and tdLN exhibit enhanced CD103+ dendritic cell trafficking. Moreover, bystander activation and proliferation of non-targeted CD8 T cells increase in B16-VEGFC tumors, indicating robust immune activation through antigen spreading. Collectively, our study establishes that VEGF-C is pivotal in shaping the anti-tumor immune response during immunotherapy by altering both the TME and the systemic immune response. By promoting T cell memory, boosting DC trafficking, and facilitating T cell recruitment, VEGF-C drives a more robust and sustained immune response against melanoma. Future research using photoconvertible mice will further elucidate the trafficking dynamics between the tumor and its draining lymph nodes post-immunotherapy, providing a more comprehensive understanding of VEGF-C's impact on the tumor microenvironment before and during immunotherapy. Colleen Foley, Lambert Potin, Casey Propst, Melody A. Swartz. VEGF-C remodels the tumor microenvironment during immunotherapy response in melanoma and promotes T cell memory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB075.

Abstract High vascular endothelial growth factor-C (VEGFC) and lymphatic vessel density in the tumor microenvironment (TME) are associated with lymph node (LN) metastasis, immunosuppression, and poor prognosis in melanoma and other solid tumors. Paradoxically, high VEGF-C also renders immunotherapy more effective in mouse models and correlates with survival after immune checkpoint therapy in patients. Here, we asked how VEGF-C may alter the systemic anti-tumor immune response in melanoma to drive response to immunotherapy, focusing on immune cell trafficking and memory T cell development during immunotherapy response. We used a B16F10 tumor model transduced to either overexpress VEGFC (B16-VEGFC) or a control vector (B16-Control) with a model of immunotherapy using transferred antigen-specific pmel CD8+ T cells activated in vivo by gp100 peptide and CpG adjuvant vaccination. We found that immunotherapy resulted in increased antigen-specific T cells in the B16-VEGFC vs. B16-Control tumors, and these antigen-specific T cells were more central memory. We also found more bystander activation and proliferation of endogenous CD8 T cells. In the B16-VEGFC tumor-draining LN, we observed increased CD103+ DC trafficking. Overall, VEGFC tumors promote more prolonged immunity after immunotherapy by supporting T cell memory development and enhancing ongoing immune activation and recruitment through increased DC trafficking to the LN and recruitment of newly activated and circulating T cells.

Background Vascular endothelial growth factor-C (VEGF-C) expression and subsequent lymphangiogenesis in the tumor microenvironment are associated with metastasis and poor prognosis in melanoma and other solid tumors. We previously demonstrated that while elevated VEGF-C promotes a highly immunosuppressive tumor microenvironment (TME), it paradoxically renders immunotherapy more effective in mouse models and correlates with improved survival after checkpoint blockade therapy in patients, a phenomenon we termed lymphangiogenic potentiation.1 This potentiation correlated with VEGFC-driven differences in the TME prior to immunotherapy, including increased recruitment of naïve T cells and cross-presenting CD103+ DCs in VEGFC-overexpressing tumors, setting up the TME for local T cell activation . Other than altering the TME, tumor lymphangiogenesis may alter the systemic immune response because of increased drainage of tumor-secreted factors, which may include both immunogenic and suppressive factors, to the tumor-draining lymph node (tdLN). Here, we asked whether and how these changes may contribute to the overall immune response as well as how the anti-tumor immune response can alter the TME in melanoma. Methods We used a B16F10 tumor model transduced to either overexpress VEGFC (B16-VEGFC) or a control vector (B16-Control) together with two different models of immunotherapy: (i) adoptive T cell transfer and (ii) peptide vaccination using transferred naïve antigen-specific pmel CD8+ T cells for in vivo tracking after activation by gp100 peptide and CpG adjuvant. Results We found that immunotherapy resulted in an increase targeted antigen-specific T cells in the B16-VEGFC vs. B16-Control tumors, including more proliferating effector cells as well as more central memory and T progenitor exhausted cells. We also found more bystander activation and proliferation of non-targeted, endogenous CD8 T cells. This was accompanied by an increase in CXCL9 in the TME of B16-VEGFC, which recruited more activated T cells from circulation. In the B16-VEGFC tdLN, we observed increased in CD103+ DC trafficking along with higher fractions of central memory T cells in both the CD4 and CD8 compartments. Conclusions Together, these results highlight the multitude of ways that tumor lymphangiogenesis can prime anti-tumor immunity and alter not only the tumor microenvironment, but also the systemic immune response by shaping the immune microenvironment of the tumor draining lymph node. VEGFC tumors promote more prolonged immunity after immunotherapy by supporting T cell memory development and enhancing ongoing immune activation and recruitment through increased DC trafficking to the lymph node and increased recruitment of newly activated and circulating T cells. Reference Fankhauser M. Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma. Sci. Transl. Med. 2017;9:eaa14712.

Lymphatic endothelial cells (LECs) express MHC class II (MHC-II) upon IFN-γ stimulation, yet recent evidence suggests that LECs cannot activate naive or memory CD4+ T cells. In this article, we show that IFN-γ-activated human dermal LECs can robustly reactivate allogeneic human memory CD4+ T cells (hCD4+ TMs), but only when TGF-β signaling is inhibited. We found that in addition to upregulating MHC-II, IFN-γ also induces LECs to upregulate glycoprotein A repetitions predominant, which anchors latent TGF-β to the membrane and potentially inhibits T cell activation. Indeed, hCD4+ TM proliferation was substantially increased when LEC-CD4+ TM cultures were treated with a TGF-β receptor type 1 inhibitor or when glycoprotein A repetitions predominant expression was silenced in LECs. Reactivated hCD4+ TMs were characterized by their proliferation, CD25 expression, and cytokine secretion. CD4+ TM reactivation was dependent on LEC expression of MHC-II, confirming direct TCR engagement. Although CD80 and CD86 were not detected on LECs, the costimulatory molecules OX40L and ICOSL were upregulated upon cytokine stimulation; however, blocking these did not affect CD4+ TM reactivation by LECs. Finally, we found that human dermal LECs also supported the maintenance of Foxp3-expressing hCD4+ TMs independently of IFN-γ-induced MHC-II. Together, these results demonstrate a role for LECs in directly modulating CD4+ TM reactivation under inflammatory conditions and point to LEC-expressed TGF-β as a negative regulator of this activation.

Abstract Growth and remodeling of tumor-associated lymphatics, termed lymphangiogenesis, has been known to increase metastasis rate in cancer patients. However, a recent study from our group has shown that tumor-associated lymphatics can improve anti-tumor immunity in melanoma upon treatment with immunotherapies. The role of lymphangiogenesis in shaping anti-tumor immune responses in breast cancer, however, remains unexplored. Furthermore, recently approved checkpoint inhibitor immunotherapies have had limited success in breast cancer patients. A better understanding of the role of tumor-associated lymphatics in shaping anti-tumor immune responses could lead to more informed decisions when treating breast cancer patients with immunotherapies. In this study, we investigated the role of induced tumor lymphangiogenesis in modulating responsiveness to different immunotherapies in a murine triple-negative mammary carcinoma model. Spontaneously metastatic 4T1 tumor cells were transduced to over-express the pro-lymphangiogenic growth factor VEGF-C (VC) to induce tumor lymphangiogensis in vivo. Flow cytometry and immunohistochemistry analyses confirmed that VEGF-C over-expressing tumors have an increase in tumor-associated lymphatics than control ones. Higher infiltrations of conv CD4 T cells and macrophages were observed in lymphangiogenic 4T1 tumors. Lymphangiogenic tumors treated with aPD-1 and aCTLA-4 checkpoint inhibitors, or an agonist of the Stimulator of Interferon Genes (STING) pathway, showed improved responsiveness to the therapy compared to the control tumors, as indicated by a reduced tumor growth rate. Moreover, positive correlations between VEGF-C expression and CD4 T cell and macrophage gene expressions were observed in primary human triple-negative breast tumor samples from The Cancer Genome Atlas (TCGA). In conclusion, our study shows that increase in tumor-associated lymphatics in triple-negative breast cancer enhances responsiveness to immunotherapies. Citation Format: Peyman Hosseinchi, Aslan Mansurov, Léa Maillat, Lambert Potin, Gavin Fujimori, Jeffrey A. Hubbell, Melody A. Swartz. Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5695.

In allergic airway inflammation, VEGFR-3-mediated lymphangiogenesis occurs in humans and mouse models, yet its immunological roles, particularly in adaptive immunity, are poorly understood. Here, we explored how pro-lymphangiogenic signaling affects the allergic response to house dust mite (HDM). In the acute inflammatory phase, the lungs of mice treated with blocking antibodies against VEGFR-3 (mF4-31C1) displayed less inflammation overall, with dramatically reduced innate and T-cell numbers and reduced inflammatory chemokine levels. However, when inflammation was allowed to resolve and memory recall was induced 2 months later, mice treated with mF4-31C1 as well as VEGF-C/-D knockout models showed exacerbated type 2 memory response to HDM, with increased Th2 cells, eosinophils, type 2 chemokines, and pathological inflammation scores. This was associated with lower CCL21 and decreased TRegs in the lymph nodes. Together, our data imply that VEGFR-3 activation in allergic airways helps to both initiate the acute inflammatory response and regulate the adaptive (memory) response, possibly in part by shifting the TReg/Th2 balance. This introduces new immunomodulatory roles for pro-lymphangiogenic VEGFR-3 signaling in allergic airway inflammation and suggests that airway lymphatics may be a novel target for treating allergic responses.

Abstract During cancer development, growth and remodeling of lymphatic vessels—termed lymphangiogenesis—is associated with increased metastasis rate. However, our group has recently shown that lymphangiogenesis can also increase tumor immune infiltration and subsequently enhance the responsiveness to immunotherapies in melanoma. However, the role of lymphangiogenesis in antitumor immunity in breast cancer remains unexplored. Moreover, despite the success of immunotherapies in different cancer types, their translation into the treatment of breast cancer has remained challenging. Here, we investigated the role of tumor lymphangiogenesis in immune cell infiltration and responsiveness to immunotherapy in a murine triple-negative breast tumor model. Spontaneously metastatic 4T1 tumor cells were transduced to overexpress the pro-lymphangiogenic growth factor VEGF-C to model tumor lymphangiogensis in vivo. We confirmed that VEGF-C overexpressing tumors were more lymphangiogenic than control ones, as assessed by flow cytometry and immunohistochemistry analyses. As previously reported, tumor VEGF-C signaling also increased the metastatic burden in the lungs. Crucially, lymphangiogenic tumors were infiltrated with increased levels of immune cells, including CD4 T cells and macrophages. Upon treatment with an agonist of the stimulator of interferon genes (STING) pathway, lymphangiogenic 4T1 tumors responded more favorably to the therapy compared to the control tumors, as indicated by a reduced tumor growth in the VEGF-C group. Furthermore, gene expression analysis of human triple-negative breast tumor samples from The Cancer Genome Atlas revealed positive correlations between VEGFC expression and CD4 T-cell and macrophage gene expressions. Taken together, these data suggest that in triple-negative breast cancer, similarly to melanoma, VEGF-C signaling is beneficial to the primary tumor microenvironment upon combination with proper immunotherapy despite promoting metastasis in untreated tumors. Citation Format: Peyman Hosseinchi, Lambert Potin, Aslan Mansurov, Léa Maillat, Jeffrey A. Hubbell, Melody A. Swartz. The immunomodulatory roles of tumor-associated lymphatics in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B97.

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in ‘immunologically cold’ tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD–IL-12) in mice bearing aggressive mouse tumours, CBD–IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD–IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD–IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD–IL-12 may potentiate CPI immunotherapy for immunologically cold tumours. A collagen-binding interleukin-12 formulation intravenously injected into mice bearing established immunologically cold mouse tumours led to marked tumour remission, particularly when combined with checkpoint-inhibitor immunotherapy.

Prior to metastasis, tumor-draining lymph nodes and distant organs are altered by a number of tumor-derived factors that help to both suppress host immunity as well as form an environment that supports metastatic tumor cells, i.e., a pre-metastatic niche. Recent studies suggest that pre-metastatic niches are induced by vesicles, including exosomes, secreted by tumor cells and cells of the tumor microenvironment. Here, we investigated the contribution of lymphatic vessels, particularly the lymphatic endothelial cells (LECs), in the active regulation of tumor exosome production and trafficking. To track exosomes, we purified and fluorescently labeled exosomes from in vitro cultures of the mouse melanoma cell line B16 F10 and of the human melanoma cell line Me260LN. Upon injection into the mouse ear dermis, fluorescent exosomes were observed within minutes in draining lymphatics, but not in surrounding blood vessels. Using the B16 F10 melanoma model overexpressing the lymphangiogenic growth factor VEGF-C, we found that upon intratumoral injection of exosomes, LECs within the tumor microenvironment abundantly take up exosomes, and at higher levels than blood endothelial cells, delivering exosomes to draining lymph nodes and eventually to the blood. In transgenic mice lacking dermal lymphatics, exosomes injected intratumorally were absent in the lymph nodes and present in far lower amounts in the blood compared to those injected into wild type mice. Additionally, while the density of endogenous blood exosomes increased upon tumor growth in wild type mice, no significant change in blood exosome density was observed in transgenic mice. Using an in vitro model where LECs are seeded onto porous inserts, we found that LEC transport of tumor exosomes occurs preferentially from their basal to apical side, and that some of these exosomes were taken up and stored intracellularly. Together, these findings suggest that lymphatic vessels constitute a major route of tumor exosome distribution to the systemic circulation and that LECs actively regulate exosome transport by active uptake.Citation Format: Lea Maillat, Lambert Potin, Witold W. Kilarski, Melody A. Swartz. Lymphatic vessels regulate exosome trafficking from tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4520.

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137(+) and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response.

Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T-lymphocyte antigen 4 antibody (CTLA4) + anti-programmed death-ligand 1 antibody (PD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors, for example 57% of total injected dose depositing in an orthotopic breast tumor model. The CBD was observed to localize throughout the tumor stroma, not merely in the subendothelial space. CBD conjugation or fusion decreased the systemic toxicity of both CTLA4+PD-L1 combination therapy and IL-2, for example eliminating hepatotoxicity with the CPI molecules and ameliorating capillary leak syndrome and pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 significantly suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells; increases in the ratio of effector CD8+ T cells to T regulatory cells were observed. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9/13 animals with the CBD-modified drugs, whereas it did so in only 1/13 animals with the unmodified drugs. Thus, the A3 domain of von Willebrand factor can be used to engineer immunotherapies with high translational promise as systemically-administered tumor targeting drugs with improved safety and efficacy compared to their native forms. The targeting approach exploits vascular permeability in the tumor to render the ubiquitous extracellular matrix protein accessible in the tumor, while sparing most other tissues. Citation Format: Jun Ishihara, Ako Ishihara, Koichi Saskai, Steve Seung-Young Lee, Mariko Yasui, Hiroyuki Abe, Lambert Potin, Peyman Hosseinchi, Kazuto Fukunaga, Michal M. Raczy, Laura T. Gray, John-Michael Williford, Masashi Fukayama, Tiffany M. Marchell, Aslan Mansurov, Aaron T. Alpar, Stephen J. Kron, Melody Swartz, Jeffrey A. Hubbell. Using matrix protein affinity to modulate the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA07.

An engineered cancer immunotherapy using a collagen-binding domain enhances efficacy and reduces adverse events.

CD40 is an immune costimulatory receptor expressed by antigen-presenting cells. Agonistic anti-CD40 antibodies have demonstrated considerable antitumor effects yet can also elicit serious treatment-related adverse events, such as liver toxicity, including in man. We engineered a variant that binds extracellular matrix through a super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144) to enhance anti-CD40′s effects when administered locally. Peritumoral injection of PlGF-2123-144-anti-CD40 antibody showed prolonged tissue retention at the injection site and substantially decreased systemic exposure, resulting in decreased liver toxicity. In four mouse tumor models, PlGF-2123-144-anti-CD40 antibody demonstrated enhanced antitumor efficacy compared with its unmodified form and correlated with activated dendritic cells, B cells, and T cells in the tumor and in the tumor-draining lymph node. Moreover, in a genetically engineered BrafV600E βCatSTA melanoma model that does not respond to checkpoint inhibitors, PlGF-2123-144-anti-CD40 antibody treatment enhanced T-cell infiltration into the tumors and slowed tumor growth. Together, these results demonstrate the marked therapeutic advantages of engineering matrix-binding domains onto agonistic anti-CD40 antibody as a therapeutic given by tumori-regional injection for cancer immunotherapy. Implications: Extracellular matrix-binding peptide conjugation to agonistic anti-CD40 antibody enhances antitumor efficacy and reduces treatment-related adverse events. Mol Cancer Ther; 17(11); 2399–411. ©2018 AACR.

Immune checkpoint inhibitors (CPI) and co-stimulatory agonist antibody therapies have exhibited considerable antitumor effects, but instances of severe side effects have been previously reported. We hypothesized that conjugation of an extracellular matrix (ECM)-binding peptide derived from placenta growth factor-2 (PlGF-2123-144) to CPI (combination of anti-CTLA4 antibody (αCTLA4) and αPD-L1) or agonistic αCD40 would accomplish local cancer immunotherapy, minimizing the antibodies9 systemic exposure. Here, we show that enhanced tissue retention and lower antibody concentrations in blood plasma resulted from PlGF-2123-144 conjugation when the antibodies were administered peri-tumorally compared to unmodified antibodies administered by the same route at the same dose. This resulted in decreases in systemic cytokine release in the blood serum, and in liver tissue damage, as was assessed both biochemically and histologically, indicating lower systemic side effects after CPI and αCD40 therapies. Particularly, we show that PlGF-2123-144 conjugation reduces the risks of αPD-L1-induced autoimmune diabetes in the male nonobese diabetic mouse. Because PD-1/PD-L1-inhibition induced diabetes has been reported in the clinic, this supports the idea that PlGF-2123-144 conjugation to antibodies may reduce adverse events. This observation with localized PlGF-2123-144-antibodies could be an important breakthrough in treating patients who have discontinued immunotherapy due to the associated side effects. Regarding efficacy, we show that either PlGF-2123-144-CPI treatment or PlGF-2123-144-αCD40 treatment showed significantly higher antitumor activity compared to unmodified forms in multiple cancer models: B16F10 and Tyr:Cre-ER+/LSL-BrafV600E/Ptenfl/fl melanoma, CT26 colon carcinoma, and MMTV-PyMT breast cancer. Local injections of PlGF-2123-144-CPI increased the number of activated CD8+ and CD4+ T cells within the tumor, compared to normal CPI. PlGF-2123-144-αCD40 treatment increased the frequency of activated dendritic cells in the tumor-draining lymph node, activated CD8+ T cells within the tumor, and secretion of endogenous antibodies against tumor cells. This immune cell activation resulted in growth suppression of a distant tumor. These data suggest the feasibility of treating patients with oligometastatic tumors by administration of PlGF-2123-144- PlGF-2123-144-CPI or PlGF-2123-144-αCD40 into one accessible metastasis. Our data suggest that local injection of either PlGF-2123-144-CPI or PlGF-2123-144-αCD40 efficiently activates tumor antigen-specific T cells while maintaining systemic immune homeostasis by avoiding influencing nontumor antigen-specific T cells. This simple approach of engineered ECM-binding immunotherapy antibodies may be clinically useful. Citation Format: Jun Ishihara, Ako Ishihara, Kazuto Fukunaga, Lambert Potin, Peyman Hosseinchi, Melody A. Swartz, Jeffrey A. Hubbell. Extracellular matrix-binding immunotherapies show enhanced antitumor efficacy and reduced adverse events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 959.

Introduction Tumour lymphangiogenesis correlates with poor prognosis and metastasis in melanoma and other cancers, yet its functional roles in regulating antitumor immunity have remained largely unexplored. Recently, we showed that despite its pro-metastatic effects, tumour lymphangiogenesis potentiates immunotherapies in melanoma. Here, we sought to further explore mechanisms of potentiation of immunotherapies and develop means to exploit the lymphatic growth factor VEGF-C therapeutically. Material and methods For this study, we used two mouse melanoma models: the injected B16F10 and the BrafV600E-PTEN-/- (±β-CAT-STA) autochthonous models. Lymphangiogenesis was inhibited using VEGFR-3 blocking antibodies, or promoted through either1 viral transduction of B16F10 with VEGF-C or2 intratumoral injections of a matrix-binding form of VEGF-C. Immunotherapies included PD-1 blockade, immune adjuvants (CpG-B and Poly I:C), or adoptive T cell transfer. For mechanistic studies, we used Batf3-/- mice, which lack cross-presenting dendritic cells (DCs) and fail to mount a potent antitumor CD8+ T cell response. Moreover, using gene expression analysis from The Cancer Genome Atlas (TCGA) database, we correlated VEGFC expression with the expression of genes associated with cross-presenting DCs such as BATF3, IRF8 and ZBTB46. Results and discussions In addition to reducing T cell infiltrates, VEGFR3 blockade in both models of melanoma reduced the number of cross-presenting DCs. Moreover, in tumours with low T cell infiltrates (coldtumours), and thus unresponsive to immunotherapies, the intratumoral delivery of matrix-binding VEGF-C increased T cell infiltration and restored the ability to respond to immunotherapies. Lymphangiogenic potentiation of immunotherapies was not observed in Batf3-/- mice, indicating the importance of cross-presenting dendritic cells and subsequent generation of tumor-specific endogenous CD8+ T cells. Finally, in the TCGA database, we found that VEGFC expression correlated with genes associated with cross-presenting DCs in human primary melanomas. Conclusion Our findings indicate that tumour lymphangiogenesis promotes recruitment of T cells and DCs into the microenvironment. By using an engineered variant of VEGF-C, we were able to transform the tumour microenvironment from a cold one to a hot one, thereby restoring responsiveness to immunotherapies. Further studies are needed to further develop this therapeutic approach.

Tumor-associated lymphangiogenesis is well known to promote metastasis and to correlate with poor prognosis in melanoma and other cancers, yet its functional roles in antitumor immunity had remained largely unexplored. In a recent study, we investigated the role of the vascular endothelial growth factor C (VEGF-C)—the main driver of lymphangiogenesis—in regulating antitumor immune response. We showed that in the B16 F10 injectable melanoma model, VEGF-C inhibition prevented T cells from infiltrating the tumor and reduced the efficacy of immunotherapies. Here, we sought to extend this concept to exploit the immunologic benefits of VEGF-C for therapeutic applications. We engineered a VEGF-C variant with an extracellular matrix-binding domain (MB-VEGF-C) allowing retention into the microenvironment after intratumoral injection. MB-VEGF-C induced increased lymphatic vasculature in tumors without reducing tumor growth rate. In an autochthonous melanoma model with reduced immune infiltrates, MB-VEGF-C intratumoral injections promoted the infiltration of cross-presenting DCs, as well as CD8+ and CD4+ T cells. Crucially, in these tumors that are normally unresponsive to immune checkpoint blockade (ICB), we could restore the responsiveness to ICB by delivering MB-VEGF-C together with immune adjuvants. Taken together, our findings suggest that VEGF-C and tumor-associated lymphangiogenesis, albeit promoting metastasis, can also be exploited to promote a T cell-inflamed microenvironment and to subsequently potentiate immunotherapies. Citation Format: Lambert Potin, Lea Maillat, Priscilla S. Briquez, Jeffrey A. Hubbell, Melody A. Swartz. Inducing therapeutic lymphangiogenesis for potentiating cancer immunotherapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1750.

Immune checkpoint blockade exhibits considerable antitumor activity, but previous studies have reported instances of severe treatment-related adverse events. We sought to explore local immune checkpoint blockade, with an antibody (Ab) form that would be retained intra- or peritumorally, limiting systemic exposure. To accomplish this, we conjugated the checkpoint blockade Abs to an extracellular matrix (ECM)-super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144). We show enhanced tissue retention and lower Ab concentrations in blood plasma after PlGF-2123-144 conjugation, reducing systemic side effects such as the risk of autoimmune diabetes. Peritumoral injections of PlGF-2123-144-anti-CTLA4 (cytotoxic T lymphocyte antigen 4) and PlGF-2123-144-anti-PD-L1 (programmed death ligand 1) Abs delayed tumor growth and prolonged survival compared to the unmodified Abs in genetically engineered murine tumor models of melanoma and breast cancer. The PlGF-2123-144-Abs increased tumor-infiltrating activated CD8+ and CD4+ T cells, resulting in a delay of distant tumor growth as well. This simple and translatable approach of engineered ECM-binding Abs may present a viable and safer approach in checkpoint blockade.

Lymphatic vessels are the primary route of communication from peripheral tissues to the immune system; as such, they represent an important component of local immunity. In addition to their transport functions, new immunomodulatory roles for lymphatic vessels and lymphatic endothelial cells have come to light in recent years, demonstrating that lymphatic vessels help shape immune responses in a variety of ways: promoting tolerance to self-antigens, archiving antigen for later presentation, dampening effector immune responses, and resolving inflammation, among others. In addition to these new biological insights, the growing field of immunoengineering has begun to explore therapeutic approaches to utilize or exploit the lymphatic system for immunotherapy.