Elena Burova

VEGF-Trap: A VEGF blocker with potent antitumor effects

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.

Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells

Angiopoietin (Ang)-2, a context-dependent agonist/antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase/Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2/Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.

Characterization of the anti-PD-1 antibody REGN2810 and its antitumor activity in human PD-1 knock-in mice

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti–PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1–dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861–70. ©2017 AACR.

Abstract 266: Antitumor activity of REGN2810, a fully human anti-PD-1 monoclonal antibody, against MC38.Ova tumors grown in immune-competent humanized PD-1 mice

Programmed Cell Death Protein 1 (PD-1), is an inhibitory checkpoint receptor, expressed in an activation-induced manner on T cells. PD-1 signals upon binding to its ligands PD-L1 and PD-L2, expressed on APCs and some types of cancer cells, and delivers inhibitory signals to T cells, promoting T-cells functional exhaustion in a tumor microenvironment. Clinically, antibodies (Abs) that block the interaction between PD-1 and PD-L1 can release the cytotoxic function of tumor-specific T cells, yielding durable objective responses in multiple cancer types. REGN2810 is a fully human hinge-stabilized IgG4P monoclonal Ab that binds to the extracellular domain of human PD-1 with high affinity and specificity. REGN2810 was selected using a combination of binding studies (Surface Plasmon Resonance [SPR]-Biacore), enzyme-linked immunosorbent assays (ELISA), novel engineered cell-based bioassays, and preclinical studies in mouse models. REGN2810 was investigated in PD-1 humanized mice, an approach that allows direct testing of clinical PD-1 Ab that does not cross-react with mouse PD-1. Humanized PD-1 mice were created by Velocigene technology to express a humanized hybrid PD-1 protein comprising the extracellular portion of human PD-1 and the transmembrane and intracellular portion of mouse PD-1. Humanized PD-1 mice were validated for inducible cell surface expression of hybrid PD-1 protein on activated T-cells, intact PD-1/PD-L1 signaling and immune responses. A weakly immunogenic murine colorectal cancer cell line MC38 was engineered to express ovalbumin (MC38.Ova) in order to increase tumor immunogenicity and allow monitoring of T-cell immune responses to well-defined antigenic ovalbumin peptides. Prophylactic treatment with REGN2810 PD-1 Ab significantly suppressed growth of subcutaneous syngeneic MC38.Ova tumors in humanized PD-1 mice in a dose dependent manner, and improved mouse survival. Therapeutic treatment with REGN2810 also promoted dose dependent regression of established MC38.Ova tumors in humanized PD-1 mice. Treatment with REGN2810 induced CD8 T cells proliferation and cytokine production in spleens of tumor bearing huPD-1 mice. The mechanism of action and robust anti-tumor efficacy of REGN2810 support its clinical development for the treatment of human cancers. Citation Format: Elena Burova, Omaira Allbritton, William Poueymirou, Venus Lai, Janelle White, Dimitris Skokos, Nicholas Papadopoulos, Drew Murphy, Israel Lowy, Ella Ioffe, Gavin Thurston. Antitumor activity of REGN2810, a fully human anti-PD-1 monoclonal antibody, against MC38.Ova tumors grown in immune-competent humanized PD-1 mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 266. doi:10.1158/1538-7445.AM2015-266

Abstract B113: In vivo characterization of anti-PD-1 antibody REGN2810 in human PD-1 knock-in mice

The Programmed Death-1 (PD-1) receptor is an immunologic inhibitory checkpoint, expressed by activated T cells. PD-1 signals upon binding to its ligands PD-L1 and PD-L2 expressed on antigen presenting cells and some types of cancer cells, and delivers inhibitory signals to T cells, resulting in suppression of T-cell effector function. In cancer patients, PD-1 is expressed on tumor-infiltrating lymphocytes and inhibits antitumor immune responses through ligand binding. Clinically, it has been shown that antibodies (Abs) that block the interaction between PD-1 and PD-L1 can release the cytotoxic function of tumor-specific T cells, yielding durable objective responses in multiple cancer types. This study describes preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4 monoclonal Ab that binds to the extracellular domain of human PD-1 with high affinity and specificity and inhibits interaction of PD-1 with its ligands. Taking advantage of the ability of human PD-1 to interact with murine PD-L1, we generated a mouse with human PD-1 gene knock-in allowing direct testing of our anti-human PD-1 Ab. Human PD-1 knock-in mice express a hybrid protein containing the extracellular portion of human PD-1, and transmembrane and intracellular domains of mouse PD-1. We demonstrated functional PD-1/PD-L1 signaling and immune responses in this model, and confirmed REGN2810 binding to hybrid PD-1 receptor on mouse T cells in vivo following REGN2810 injections. Prophylactic and therapeutic treatments of subcutaneous syngeneic tumors with REGN2810 in human PD-1 knock-in mice resulted in a dose-dependent suppression of tumor growth. Currently REGN2810 anti-PD-1 Ab is in phase 1 clinical trials for oncology indications. Citation Format: Elena Burova, Omaira Allbritton, William Poueymirou, Venus Lai, Janelle Waite, Dimitris Skokos, Nicholas Papadopoulos, Drew Murphy, Israel Lowy, Ella Ioffe, Gavin Thurston. In vivo characterization of anti-PD-1 antibody REGN2810 in human PD-1 knock-in mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B113.

Abstract B129: Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion

Immune checkpoint blockade has revolutionized cancer immunotherapy, and combination treatment with antibodies against multiple inhibitory receptors promises to improve efficacy substantially. However, the spatiotemporal and combinatorial expression profile of various checkpoint receptors on effector T cells and the effect of combined blockade on immune function remain poorly defined. In this study we set out to determine the co-expression kinetics and functional impact of multiple checkpoint receptors (PD-1, LAG-3, TIM-3, and BTLA) on tumor antigen-specific effector T cells in controlled in vitro culture systems and in the tumor environment. To elicit tumor antigen-specific responses we immunized mice with irradiated MC38 tumor cells engineered to express chicken ovalbumin (OVA; MC38.OVA) and used pentamer reagents to identify OVA257-264-specific CD8 T cells. Repeated stimulation with irradiated MC38.OVA cells or irradiated OVA-pulsed splenocytes ex vivo resulted in robust proliferation and activation of antigen-specific T cells as determined by BrdU incorporation and production of IFN-γ. Most OVA-specific cells expressed PD-1 ex vivo and maintained PD-1 expression during repeated cycles of in vitro stimulation. In contrast, antigen-specific T cells did not express TIM-3 or LAG-3 ex vivo, yet both receptors were induced on the majority of PD1 positive, but not PD-1 negative, cells upon repeated stimulation ex vivo, resulting in double and triple positive cells. The frequency of BTLA expressing cells remained low under all conditions. The addition of PD-1 and TIM-3 blocking antibodies during ex vivo restimulation enhanced T cell activation, demonstrating that these receptors mediate inhibitory function. Consistent with our in vitro data demonstrating that repeated antigen stimulation promotes the coordinated expression of checkpoint receptors, we found that the vast majority of MC38.OVA or Colon26 tumor infiltrating T cells (TILs) express PD-1, and most PD-1 positive cells also co-express TIM-3 and LAG-3, whereas these receptors are largely absent from T cells in secondary lymphoid organs. Importantly, combination treatment of tumor-bearing mice with anti-PD-1 and anti-TIM-3, or anti-PD-1 and anti-LAG-3 antibodies enhanced the immune control of tumor growth in both models as compared to the respective monotherapies. Finally, in vitro activation of human PBMC-derived T cells resulted in a similar sequential upregulation of checkpoint inhibitors ultimately resulting in their co-expression. Here, we have established a culture system to delineate the hierarchical expression of inhibitor checkpoint receptors on tumor antigen-specific murine T cells during repeated antigen stimulation. Defining the inhibitory checkpoint receptor landscape will guide the process of identifying the most promising combinations of checkpoint blockers in cancer immunotherapy. Citation Format: Robert J. Durso, Jerry Pei, Michelle Russell, Pratha Budhani, Elena Burova, Chandrika Taduriyasas, Ella Ioffe, Markus Mohrs, Gavin Thurston, Jie Dai. Multiple immune checkpoint receptors are co-expressed on tumor antigen-specific T cells and contribute to tumor immune evasion [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B129.

Abstract 1484: Combined treatment with anti-LAG-3 and anti-PD-1 fully human monoclonal antibodies inhibits tumor growth in immunocompetent double-humanized LAG-3/PD-1 mice

Lymphocyte-activation gene 3 (LAG-3) receptor is expressed on activated CD4 and CD8 T cells, γδ T cells, Treg, NK, NKT, B and plasmacytoid dendritic cells. LAG-3 binds to major histocompatibility complex class II (MHC II) and delivers inhibitory signals that regulate T cell proliferation and cytokine production. LAG-3 blockade augments T cell proliferation and activation. Programmed cell death 1 (PD-1) receptor upon binding to its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) also delivers an inhibitory checkpoint signal that is critical for the establishment and maintenance of peripheral T cell tolerance. PD-1 signaling plays a critical role in the tumor microenvironment by allowing tumors to escape immune surveillance. We tested in vivo activity of anti-mouse PD-1 and anti-mouse LAG-3 antibodies in several preclinical syngeneic tumor models and showed that combination treatment with both antibodies resulted in an additive anti-tumor effect compared to either single antibody treatment. TaqMan analysis in the MC38 tumor model demonstrated CD8+ T cell expansion in both the draining lymph nodes and spleens of mice in the combination treatment group. Anti-human LAG-3 antibody is a fully human monoclonal antibody developed for cancer immunotherapy. It binds with high affinity to human LAG-3 and blocks LAG-3/MHC II interaction. Fully human monoclonal antibody REGN2810 binds with high affinity to human PD-1 and blocks PD-1 interaction with PD-L1 and PD-L2. Double humanized LAG-3/PD-1 mice were engineered using VelociGene® technology to replace the extracellular domains of mouse Pdcd1 and Lag3 genes with the corresponding regions of human PD-1 and human LAG-3 genes. To validate humanized protein expression, we examined PD-1 and LAG-3 protein expression on T cells after anti-CD3/anti-CD28 antibody stimulation. We confirmed binding of human LAG-3 and PD-1 to the corresponding mouse ligands by cell adhesion assay for human LAG-3 and mouse MHC II interactions and by SPR-Biacore for human PD-1 and mouse PD-L1 interactions, respectively. Combination of REGN2810 and anti-hLAG-3 antibodies in MC38.ova tumor model in double humanized LAG-3/PD-1 mice, which allows testing of clinical antibodies that do not cross to mouse receptors, demonstrated improved efficacy, including reduced tumor growth and improved survival, compared to REGN2810 and anti-hLAG-3 monotherapies. Robust anti-tumor efficacy of REGN2810 and anti-hLAG-3 combination in preclinical setting supports their clinical development as a combination cancer immunotherapy. Citation Format: Elena Burova, Omaira Allbritton, Chandrika Taduriyasas, Venus Lai, William Poueymirou, Nicholas Papadopoulos, Douglas MacDonald, William Olson, Markus Mohrs, Ella Ioffe, Gavin Thurston. Combined treatment with anti-LAG-3 and anti-PD-1 fully human monoclonal antibodies inhibits tumor growth in immunocompetent double-humanized LAG-3/PD-1 mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1484.

Abstract 5035: Blockade of VEGF with ziv-aflibercept (VEGF Trap) enhances anti-tumor efficacy of CTLA-4 blocking antibody in an Fc dependent manner

VEGF-dependent angiogenesis is required for tumor progression. Ziv-aflibercept (VEGF Trap) is a VEGF inhibitor, which exhibits potent antitumor activity in preclinical models and was approved by the FDA in combination with FOLFIRI for patients with metastatic colorectal cancer (mCRC) who did not respond or progressed following an oxaliplatin-containing regimen. Recent data suggest that VEGF blockade may also modulate tumor immunity by promoting necrosis and inflammation, however the precise mechanism is not fully understood. CTLA-4 blockade promotes tumor immunity by, in part, reversing suppressive effects of CTLA-4 receptor on T cell activation, and exhibits synergistic effects with other immunomodulatory agents in both clinical and experimental settings. In this study, we examined the combination of ziv-aflibercept (at 10 mg/kg) and CTLA-4 blocking antibody (at 200 ug/mouse) administered five times within two weeks in two mouse colon carcinoma models (Colon26 and MC38). Prophylactic therapy with either single agent inhibited Colon26 tumor growth. Responses to ziv-aflibercept were associated with reduction of tumor vascularity, while those to α-CTLA-4 murine IgG2b antibody were associated with enrichment of tumor CD8, but not CD4, T cells as measured by flow cytometry. Combination therapy resulted in complete tumor regression and significantly improved survival (0%, 30% and 70% survival in ziv-aflibercept, α-CTLA-4 and combination groups respectively). Pathological evaluation of ziv-aflibercept treated tumors by histostaining revealed increased tumor necrosis, which became more severe in the combination group. α-CTLA-4 antibody did not promote definitive tumor necrosis, but resulted in increased immune cell infiltrate composed mainly of lymphocytes and macrophages. To address the role of the immunoglobulin constant region of α-CTLA-4 antibody, we compared the effect of murine IgG2a and IgG2b antibody versions with high and low effector function respectively. α-CTLA-4 IgG2a antibody was significantly more potent than IgG2b, resulting in eradication of established Colon26 tumors and partial growth inhibition of established MC38 tumors, whereas these established tumors were resistant to α-CTLA4 IgG2b therapy. Co-administration of ziv-aflibercept significantly improved α-CTLA4 IgG2a antibody efficacy against established MC38 tumors. These results suggest that combining immunotherapy and ziv-aflibercept may be beneficial for the treatment of established tumors in a clinical setting. Citation Format: Elena Burova, Ella Ioffe, Chandrika Taduriyasas, Omaira Allbritton, Gaurav Tyagi, Lekan Oyejide, John Rudge, Israel Lowy, Gavin Thurston. Blockade of VEGF with ziv-aflibercept (VEGF Trap) enhances anti-tumor efficacy of CTLA-4 blocking antibody in an Fc dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5035. doi:10.1158/1538-7445.AM2014-5035

Abstract 1023: Comparison of the effects of aflibercept (VEGF Trap) and small molecule kinase inhibitors on tumors and normal organs in mouse models

Introduction: The VEGF signaling pathway is important for tumor angiogenesis, thus a number of approaches have been developed to therapeutically inhibit the pathway. Clinical benefit has been shown by both protein therapeutics that block VEGF itself, and by small molecules that target the kinase activity of VEGF receptors. However, it is not known whether these different classes of agents provide similar levels of VEGF inhibition in tumors, nor whether they produce a similar degree of perturbation to normal organs. In this study, we compared the effects of aflibercept (VEGF Trap), a protein inhibitor of VEGF, to those of the kinase inhibitors sorafenib and sunitinib on tumors and several normal organs in mouse models. Methods: Transcriptional profiles were compared from A431 human epidermoid carcinoma xenografts, as well as kidney, liver, lung and heart, following three days of treatment of mice with aflibercept (25 mg/kg, once), sunitinib (80 mg/kg, daily), or sorafenib (160 mg/kg, daily). All three agents were used at pharmacologically active doses that induce significant tumor growth inhibition. Kinase inhibitors were used at maximum dose that did not cause general toxicity such as body weight loss. RNA microarray was used to establish gene signatures, and real-time PCR was used to validate gene-specific expression levels. Results: Treatment of mice with sorafenib or sunitinib induced large-scale transcriptional changes in normal organs, including a significant overlap of the regulated genes and strong suppression of hemoglobin genes Hba and Hbb. In contrast, aflibercept treatment induced transcriptional changes in a much smaller number of genes in normal organs and had no significant effect on hemoglobin transcript levels. None of the agents changed expression of endothelial cell marker genes such as Pecam1 and Robo4 in these normal organs, indicating that VEGF inhibitors do not target stable vasculature. In tumor xenografts, aflibercept regulated a significantly larger number of stromal genes compared to either kinase inhibitor. Aflibercept, sorafenib or sunitinib suppressed expression of Pecam1 in tumors (by 76%, 56% and 21% respectively) and Robo4 (by 56%, 20% and 30% respectively), as well as VEGF regulated genes Ang2 (by 59%, 44% and 41% respectively) and Dll4 (by 78%, 35% and 64% respectively), indicating reduced tumor vessel density and inhibition of VEGF signaling. The three treatments affected a similar number of genes expressed by the tumor cells, including upregulation of hypoxia-induced genes. Conclusions: Our findings indicate that aflibercept treatment of mice induces fewer changes in gene expression in normal tissues than the kinase inhibitors sunitinib or sorafenib, while at the same time produces similar or larger reduction of the tumor vasculature and stroma genes in mouse xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1023. doi:1538-7445.AM2012-1023