Supplementary Figure S7-10 and Table S1-2

Abstract The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis and better responses to immune checkpoint blockade in many cancer types. Elucidation of the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to antitumor responses may facilitate the development of improved treatment strategies. Lymphotoxin β receptor (LTβR) signaling is a critical regulator of lymph node organogenesis and can cooperate with antiangiogenic and immune checkpoint blockade treatment to augment tumor-associated HEV formation. In this study, we demonstrated that LTβR signaling modulates the tumor microenvironment via multiple mechanisms to promote antitumor T-cell responses. Systemic activation of the LTβR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T-cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTβR agonism on DC activation and maturation and associated DC-mediated T-cell activation. Single-agent LTβR agonist treatment inhibited syngeneic tumor growth in a CD8+ T-cell–dependent and HEV-dependent manner, and the LTβR agonist enhanced antitumor effects of anti-PD-1 and CAR T-cell therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTβR agonism and lymphotoxin ⍺ expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA4 treatment. Collectively, this study highlights crucial functions of LTβR signaling in modulating the tumor microenvironment and could inform future HEV/TLS-based strategies for cancer treatments. Significance: LTβR mediates tumor-specific high endothelial venule formation and immunomodulation of the tumor microenvironment that promotes antitumor immune responses, supporting LTβR agonism as an approach to enhance the antitumor efficacy of immunotherapies.

Sezary syndrome (SS) is a rare, aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) that lacks adequate therapeutic options and representative small-animal models. Here, we demonstrate that IL-15 is a critical CTCL growth factor. Importantly, an immunodeficient knock-in mouse model genetically engineered to express human IL-15 uniquely supported the growth of SS patient samples relative to conventional immunodeficient mouse strains. SS patient-derived xenograft (PDX) models recapacitated key pathological features of the human disease, including skin infiltration and spread of leukemic cells to the periphery, and maintained the dependence on human IL-15 upon serial in vivo passaging. Detailed molecular characterization of the engrafted cells by single-cell transcriptomic analysis revealed congruent neoplastic gene expression signatures but distinct clonal engraftment patterns. Overall, we document an important dependence of Sezary cell survival and proliferation on IL-15 signaling and the utility of immunodeficient humanized IL-15 mice as hosts for SS - and potentially other T and NK cell-derived hematologic malignancies - PDX model generation. Furthermore, these studies advocate the thorough molecular understanding of the resultant PDX models to maximize their translational impact.

Legends for Supplementary Tables S1 and S2

<p>Legends for Supplementary Figures S1-S12</p>

High endothelial venules (HEV) are specialized blood vessels that mediate lymphocyte trafficking to lymph nodes. Tertiary lymphoid structures (TLS) are ectopic lymphoid formations that develop in inflamed, infected or tumoral tissues. TLS contain HEV and B cell follicles surrounded by a T-cell zone and are characterized by abundant chemokine expression. The presence of TLS and HEV in solid tumors is positively correlated with patient survival in many cancer types, and may even be predictive of better response to immune-checkpoint blockade. However, the molecular mechanisms underlying the intratumoral HEV and TLS formation remain unclear. Using murine syngeneic tumor models, we found that systemic activation of lymphotoxin beta receptor (LTBR) with an agonistic antibody induced tumor-specific HEV formation, increased dendritic cell (DC) and T cell infiltration, and enhanced T cell activation in the tumor. In vitro assays revealed that LTBR agonism directly upregulated activation and maturation markers in bone marrow-derived DCs and promoted DC-mediated CD4 and CD8 T cell activation. Single agent LTBR agonist treatment attenuated Colon26 tumor growth in a CD8 T cell-dependent manner. In combination treatment studies, LTBR agonism further augmented anti-tumor efficacy of anti-PD1 and of CAR-T therapy. Notably, LTBR agonist mAb treatment upregulated the expression of TLS-related chemokines and induced TLS-like structures in approximately 20% of treated tumors. To enhance TLS induction, we generated syngeneic tumor models engineered to express several cytokines/chemokines and performed an in vivo screen for tumor-associated TLS formation. We identified some factors that in combination with LTBR agonism induced B cell enrichment and immature TLS formation, while others promoted robust TLS induction and anti-tumor effect. TLS formation induced by combined LTBR agonism and cytokine expression is associated with augmented anti-tumor responses to anti-CTLA-4 treatment. By studying anti-tumor mechanisms of LTBR agonism-mediated HEV and TLS formation, this work informs the future therapeutic strategies to boost T cell infiltration and activation in solid tumors. Citation Format: Disi An, Guoying Chen, Wei Wang, Katja Mohrs, David DiLillo, Christopher Daly, Gavin Thurston, John Lin, Namita Gupta, Mickey Atwal, Frank Kuhnert. Boosting anti-tumor immunity by promoting high endothelial venule and tertiary lymphoid structure formation in solid tumors via LTBR agonism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4134.

<p>Supplementary Figure S1: Correlation of REGN421 serum concentrations and anti-HT1080 tumor activity Supplementary Figure S2: The potent anti-tumor activity of targeting Dll4 in ovarian xenograft models is dependent on blocking stromal Dll4 Supplementary Figure S3: Dll4 antibody REGN1035 treatment induces abnormal tumor vessels in ovarian cancer xenograft models Supplementary Figure S4: Dll4 expression in ovarian cancer xenograft models is restricted to the tumor vasculature Supplementary Figure S5: Notch receptor expression in ovarian cancer xenograft models Supplementary Figure S6: Notch signaling in TOV-112D cells is mediated predominantly by Notch1 Supplementary Figure S7: Combined blockade of Dll4 and VEGF reverses liver vascular changes induced by Dll4 blockade alone Supplementary Figure S8: Dll4 is expressed in the endothelium of adult heart and liver Supplementary Figure S9: Notch1 signaling activity in heart and liver is dependent on Dll4 Supplementary Figure S10: Pericyte coverage in ovarian xenograft tumors is unaltered in response to anti-Dll4 antibody treatment Supplementary Figure S11: Direct contact between endothelial and tumor cells in TOV-112D tumors Supplementary Figure S12: Direct contact between endothelial and tumor cells in A2780 tumors</p>

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4β7 in conventional T cells while preserving α4β7 in regulatory T cells, with findings suggesting increased β1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4β7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Supplementary Table S1: Dll4 Ab REGN421 binds human and monkey Dll4 with high affinity Supplementary Table S2: Combined blockade of Dll4 and VEGF signaling results in enhanced inhibition of A2780 ovarian tumors

Abstract The Notch ligand delta-like 4 (Dll4) has been identified as a promising target in tumor angiogenesis in preclinical studies, and Dll4 inhibitors have recently entered clinical trials for solid tumors, including ovarian cancers. In this study, we report the development of REGN421 (enoticumab), a fully human IgG1 monoclonal antibody that binds human Dll4 with sub-nanomolar affinity and inhibits Notch signaling. Administering REGN421 to immunodeficient mice engineered to express human Dll4 inhibited the growth of several human tumor xenografts in association with the formation of nonfunctional tumor blood vessels. In ovarian tumor xenograft models, Dll4 was expressed specifically by the tumor endothelium, and Dll4 blockade by human-specific or mouse-specific Dll4 antibodies exerted potent antitumor activity, which relied entirely on targeting Dll4 expressed by tumor stromal cells but not by the tumor cells themselves. However, Dll4 blockade reduced Notch signaling in both blood vessels and tumor cells surrounding the blood vessels, suggesting that endothelial-expressed Dll4 might induce Notch signaling in adjacent ovarian tumor cells. The antitumor effects of targeting Dll4 were augmented significantly by simultaneous inhibition of VEGF signaling, whereas this combined blockade reversed normal organ vascular changes induced by Dll4 blockade alone. Overall, our findings deepen the rationale for antibody-based strategies to target Dll4 in ovarian cancers, especially in combination with VEGF blockade. Cancer Res; 75(19); 4086–96. ©2015 AACR.

The Notch ligand delta-like 4 (Dll4) has been identified as a promising new target in tumor angiogenesis in preclinical studies, and Dll4 inhibitors have recently entered clinical trials for solid tumors, including ovarian cancers. We previously demonstrated that pharmacological blockade of the Dll4-Notch axis results in tumor vessel abnormalization and associated reduction of tumor growth. Using VelocImmune® mice, we identified a fully human IgG1 monoclonal antibody, REGN421 (called enoticumab), which binds human Dll4 with sub-nanomolar affinity and potently inhibits Notch signaling. Treatment of immunodeficient mice engineered to express human Dll4 with enoticumab inhibited growth of several human tumor xenografts and induced non-functional tumor blood vessels. In ovarian tumor xenograft models, Dll4 was expressed specifically by tumor endothelium, and blockade of Dll4 with either human- or mouse-specific Dll4 antibodies showed potent anti-tumor activity was entirely dependent on targeting stromal Dll4 but not tumor cell-expressed Dll4. Blockade of Dll4 reduced Notch signaling in both blood vessels and in tumor cells surrounding blood vessels, suggesting that Dll4 on endothelial cells could induce Notch signaling on ovarian tumor cells. The anti-tumor activity of Dll4 blockade in ovarian tumors was markedly augmented by simultaneous inhibition of VEGF signaling. Importantly, the combined blockade of Dll4 and VEGF reversed normal organ vascular changes that were induced by Dll4 blockade alone. These studies lend further support for therapeutic targeting of Dll4 as a promising new angiogenesis-based anticancer strategy in ovarian cancer, in particular in combination with anti-VEGF agents. Citation Format: Frank Kuhnert, Guoying Chen, Sandra Coetzee, Jessica Kirshner, Gavin Thurston. Potent antitumor activity of Dll4 blockade in ovarian xenografts mediated by blocking stromal Dll4. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A19.

Potent anti-tumor activity of blocking stromal Dll4 in ovarian xenograft models Delta-like ligand 4 (Dll4) is an emerging anticancer target given its predominant tumor vasculature expression and its role in regulating angiogenic sprouting. We have previously demonstrated that pharmacological blockade of the Dll4-Notch axis results in an excessive production of aberrant non-functional tumor vessels, and these changes were associated with reduced tumor growth. Using VelocImmune® mice, we identified a fully human IgG1 monoclonal antibody, termed REGN421/SAR153192, which binds human Dll4 and potently neutralizes Notch signaling. REGN421 treatment caused potent and dose-dependent inhibition of a number of human tumor xenografts grown in immunodeficient mice engineered to express human Dll4. In the current study, we found that REGN421 treatment of ovarian xenograft models produces potent anti-tumor effects that are dependent on targeting Dll4 in the tumor stroma as opposed to tumor cell-expressed Dll4. In particular, REGN421 treatment (2.5 mg/kg, once weekly) of humanized Dll4 mice bearing established subcutaneous TOV-112D or intraperitoneal A2780 human tumor xenografts resulted in growth inhibition of 86% and 83%, respectively. The inhibition of ovarian tumor growth by REGN421 was associated with a marked increase in tumor vascular structures but reduced vascular perfusion, consistent with the function of Dll4 as a regulator of angiogenic sprouting. Similar anti-tumor effects were observed by strictly targeting stromal Dll4 with a mouse Dll4-specific surrogate antibody REGN1035 in SCID mice bearing OVCAR3 tumors. In contrast, the specific blockade of tumor cell-expressed human Dll4 did not exhibit any appreciable anti-tumor activity, indicating the lack of tumor growth-promoting autocrine Dll4-Notch tumor cell signaling in these models. Finally, the combined treatment of Dll4 antibody with the anti-VEGF agent ziv-aflibercept (VEGF Trap) resulted in enhanced anti-tumor effects and virtually the complete suppression of intraperitoneal A2780 tumor growth, suggesting clinical benefit for the combined blockade of VEGF and Dll4 in ovarian cancer. These studies lend further support for the therapeutic targeting of Dll4 as a promising new angiogenesis-based anticancer strategy in ovarian cancer, both as a single agent and in combination with anti-VEGF agents. Citation Format: Frank Kuhnert, Guoying Chen, Gavin Thurston. Potent anti-tumor activity of blocking stromal Dll4 in ovarian xenograft models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5091. doi:10.1158/1538-7445.AM2013-5091

The essential role of the Delta‐like ligand 4 (Dll4)‐Notch signaling pathway in T‐lymphocyte development is well established. It has been shown that specific inactivation of Dll4 on thymic stromal cells during early post‐natal development leads to a deregulation in T‐cell differentiation. However, whether ongoing Dll4‐Notch signaling is required for T‐cell development in the adult thymus is unknown. The use of anti‐Dll4 Abs allowed us to confirm and expand previous studies by examining the kinetics and the reversibility of Dll4‐Notch signaling blockade in T‐cell development in adult mice. We found that anti‐Dll4 treatment reduced thymic cellularity after 7 days, as a consequence of a developmental delay in T‐cell maturation at the pro‐T‐cell double negative 1 (CD4−CD8−c‐kit+CD44+CD25−) stage, leading to decreased numbers of immature double‐positive (CD4+CD8+) T cells without affecting the frequency of mature single positive CD4+ and CD8+ thymocytes, while promoting alternative thymic B‐cell expansion. This cellular phenotype was similarly observed in both young adult and aged mice (>1.5 years), extending our understanding of the ongoing role for Dll4‐Notch signaling during T‐cell development in the adult thymus. Finally, after cessation of Dll4 Ab treatment, thymic cellularity and thymocyte subset ratios returned to normal levels, indicating reversibility of this phenotype in both adult and aged mice, which has important implications for potential clinical use of Dll4‐Notch inhibitors.