Jessica R. Kirshner

Elesclomol induces cancer cell apoptosis through oxidative stress

Elesclomol (formerly STA-4783) is a novel small molecule undergoing clinical evaluation in a pivotal phase III melanoma trial (SYMMETRY). In a phase II randomized, double-blinded, controlled, multi-center trial in 81 patients with stage IV metastatic melanoma, treatment with elesclomol plus paclitaxel showed a statistically significant doubling of progression-free survival time compared with treatment with paclitaxel alone. Although elesclomol displays significant therapeutic activity in the clinic, the mechanism underlying its anticancer activity has not been defined previously. Here, we show that elesclomol induces apoptosis in cancer cells through the induction of oxidative stress. Treatment of cancer cells in vitro with elesclomol resulted in the rapid generation of reactive oxygen species (ROS) and the induction of a transcriptional gene profile characteristic of an oxidative stress response. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of gene transcription by elesclomol. In addition, N-acetylcysteine blocked drug-induced apoptosis, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of elesclomol. Excessive ROS production and elevated levels of oxidative stress are critical biochemical alterations that contribute to cancer cell growth. Thus, the induction of oxidative stress by elesclomol exploits this unique characteristic of cancer cells by increasing ROS levels beyond a threshold that triggers cell death. [Mol Cancer Ther 2008;7(8):2319–27]

Dll4-Notch signaling as a therapeutic target in tumor angiogenesis.

Tumor angiogenesis is an important target for cancer therapy, with most current therapies designed to block the VEGF signaling pathway. However, clinical resistance to anti-VEGF therapy highlights the need for targeting additional tumor angiogenesis signaling pathways. The endothelial Notch ligand Dll4 (delta-like 4) has recently emerged as a critical regulator of tumor angiogenesis and thus as a promising new therapeutic anti-angiogenesis target. Blockade of Dll4-Notch signaling in tumors results in excessive, non-productive angiogenesis with resultant inhibitory effects on tumor growth, even in some tumors that are resistant to anti-VEGF therapies. As Dll4 inhibitors are entering clinical cancer trials, this review aims to provide current perspectives on the function of the Dll4-Notch signaling axis during tumor angiogenesis and as a target for anti-angiogenic cancer therapy.

A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys

Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies.

Ongoing Dll4-Notch signaling is required for T-cell homeostasis in the adult thymus.

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.

Dll4 Blockade in Stromal Cells Mediates Antitumor Effects in Preclinical Models of Ovarian Cancer

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.

Bispecific Antibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681–93. ©2017 AACR.

In-silico discovery of cancer-specific peptide-HLA complexes for targeted therapy

BackgroundMajor Histocompatibility Complex (MHC) or Human Leukocyte Antigen (HLA) Class I molecules bind to peptide fragments of proteins degraded inside the cell and display them on the cell surface. We are interested in peptide-HLA complexes involving peptides that are derived from proteins specifically expressed in cancer cells. Such complexes have been shown to provide an effective means of precisely targeting cancer cells by engineered T-cells and antibodies, which would be an improvement over current chemotherapeutic agents that indiscriminately kill proliferating cells. An important concern with the targeting of peptide-HLA complexes is off-target toxicity that could occur due to the presence of complexes similar to the target complex in cells from essential, normal tissues.ResultsWe developed a novel computational strategy for identifying potential peptide-HLA cancer targets and evaluating the likelihood of off-target toxicity associated with these targets. Our strategy combines sequence-based and structure-based approaches in a unique way to predict potential off-targets. The focus of our work is on the complexes involving the most frequent HLA class I allele HLA-A*02:01. Using our strategy, we predicted the off-target toxicity observed in past clinical trials. We employed it to perform a first-ever comprehensive exploration of the human peptidome to identify cancer-specific targets utilizing gene expression data from TCGA (The Cancer Genome Atlas) and GTEx (Gene Tissue Expression), and structural data from PDB (Protein Data Bank). We have thus identified a list of 627 peptide-HLA complexes across various TCGA cancer types.ConclusionPeptide-HLA complexes identified using our novel strategy could enable discovery of cancer-specific targets for engineered T-cells or antibody based therapy with minimal off-target toxicity.

ImmunoPET Imaging of Endogenous and Transfected Prolactin Receptor Tumor Xenografts

Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53-5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of ∼14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR.

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.

Abstract A19: Potent antitumor activity of Dll4 blockade in ovarian xenografts mediated by blocking stromal Dll4

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.