Heather Huet

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.

Effector-Mediated Eradication of Precursor B Acute Lymphoblastic Leukemia with a Novel Fc-Engineered Monoclonal Antibody Targeting the BAFF-R

B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia (pre-B ALL) cells, but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here, we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild-type cells, but were more sensitive to drug treatment in vitro. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti–BAFF-R monoclonal antibody optimalized for FcRγIII-mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated natural killer cell–mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti–pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted. Mol Cancer Ther; 13(6); 1567–77. ©2014 AACR.

Abstract 3853: TAS266, a novel tetrameric nanobody agonist targeting death receptor 5 (DR5), elicits superior antitumor efficacy than conventional DR5-targeted approaches

Preferential induction of apoptosis in cancer cells has been the objective of therapeutic strategies targeting apoptotic pathways. To this end, multiple therapeutic agonists of Death Receptors 4 and 5 (DR4, DR5), have been developed and are under clinical evaluation. Although these agonists, including antibodies and soluble ligand TRAIL, demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation for the discrepant pre-clinical and clinical results is that DR5 may play a more prominent role in in vitro model systems as opposed to cancers in humans. Alternatively, these results might indicate that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients. In particular, naturally dimeric antibody agonists require secondary cross-linking via Fcα receptors expressed on immune cells present in the tumor microenvironment to achieve optimal clustering of DR5 into a ternary active state. Because immune cell content in the tumor can be heterogeneous, reliance on this secondary mechanism for activity may limit the potency of these antibodies. To overcome this limitation, a novel nanobody approach was taken to eliminate the need for cross-linking and improve receptor activation with the goal of generating a significantly more potent DR5 agonist. Nanobodies are a class of therapeutic proteins based on single, high affinity heavy chain domain (VHH) antibodies that naturally occur in camelid species, and these VHH domains can be linked to form multivalent structures (di-, tri-, tetra-, etc). This approach led to the development of a tetrameric DR5 targeted agonist, TAS266, with significantly greater avidity for DR5 binding. TAS266 activates downstream caspases with more rapid kinetics and is up to 1000-fold more potent in cell death assays when compared to a cross-linked DR5 antibody or TRAIL. In vivo, TAS266 elicits single dose tumor regressions in multiple tumor xenograft models and sustained tumor regressions after treatment cessation. TAS266 showed superior anti-tumor activity compared to a DR5 agonist antibody and TRAIL, including the ability to induce tumor regression in a patient-derived primary pancreatic tumor model that is insensitive to the agonist antibody. Thus, TAS266 has the potential for superior clinical activity in settings insensitive to the conventional therapeutic approaches to DR5. First-in-man trials are expected to begin in 2012. 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 3853. doi:1538-7445.AM2012-3853

Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.

Abstract A77: Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells

HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancer diagnoses, often signifying poor prognosis. The current standard of care for HER2 amplified cancer is the HER2 targeting antibody trastuzumab with chemotherapy. In the setting of HER2 amplified breast cancer, the combination of HER2 targeting antibodies trastuzumab and pertuzumab with the chemotherapeutic docetaxel is the frontline therapy. Despite the success of targeting the HER2 pathway, there are still many patients who are refractory or relapse following HER2 targeting regimens. Outside of the setting of HER2 overexpression or gene amplification, targeting HER2-positive cancers has thus far been ineffective. Engineered autologous T-cells, including chimeric antigen receptors (CARs) and high affinity T-cell receptors (TCRs), have gained attention recently due to their potent efficacy, with overall response rates reaching 80% and examples of long lasting remissions, particularly in advanced lymphoma and leukemia. However, initial clinical attempts to target HER2-amplified breast cancer with CAR-T cell therapy met with either acute toxicities or lack of efficacy. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T cell therapy technology developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR chimeric construct is composed of the high-affinity CD16 (FCGR3A) V158 variant with the signaling domains from CD3z and 4-1BB. ACTR is designed to engage the Fc domain of human IgG1 antibodies when opsonized to the cognate target cells and deliver an activation signal to the engineered T cells. ACTR T cells combined with either HER2 targeting trastuzumab or pertuzumab exhibited potent cytotoxic activity, cytokine response and proliferation on a HER2 amplified tumor cell lines. ACTR activity was specific to antibody treated cells, and had little activity on HER2 low or negative tumor lines. Furthermore, the effectiveness of ACTR T cells in a non-amplified HER2 (HER2 low) setting was tested in the presence of a combination of trastuzumab and pertuzumab. This multi-antibody combination increased the cytotoxicity by ACTR T cells, whereas trastuzumab or pertuzumab as single antibody combinations with ACTR had little effect, suggesting a potential therapeutic approach for this disease setting with high unmet medical need. Importantly, the ACTR platform decouples the targeting moiety from the engineered T cell allowing for an antibody dose response which may spare cytotoxicity on normal cells. The specific response to HER2 positive tumor cells in the presence of trastuzumab, pertuzumab or the combination of HER2 directed antibodies demonstrates the potential therapeutic activity of ACTR T cells and supports consideration of clinical testing of ACTR T cells in HER2-positive cancers. Furthermore, combining multiple targeting antibodies to achieve greater potency and efficacy is unique to the ACTR technology and demonstrates the potential for such activity across many other tumor targets and tumor targeting antibodies. Citation Format: Casey B. Judge, Rachel DeBarge, Eugene Choi, Katie O9Callaghan, Lindsay Edwards, Birgit Schultes, Seth Ettenberg, Heather A. Huet. Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A77.

Abstract 3762: Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell

Glypican-3 (GPC3) is a GPI-anchored member of the heparan sulfate proteoglycan family. GPC3 is an oncofetal antigen expressed transiently during fetal development with re-expression during malignant transformation. GPC3 is an ideal tumor target as expression has been found in numerous epithelial malignancies, with highest expression in hepatocellular carcinoma (HCC) and non-small cell lung carcinoma (NSCLC), and normal tissue expression is highly restricted (Baumhoer D., Am J Clin Pathol, 2008.). Adoptive T-cell therapy with single-chain variable fragment (scFv)-derived chimeric antigen receptors (CARs) has transformed cancer therapy, but the broad applicability of this approach has been limited in part by safety concerns due the constitutive expression of a biologically active targeting receptor. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy designed to engage the Fc domain of therapeutic antibodies opsonized to tumor cells to mediate anti-tumor activity. ACTR activity is therefore both regulatable and flexible, providing enhanced therapeutic control and improved safety of the T cell therapy. Using both HCC and NSCLC tumor cell lines, we tested a panel of wild-type and afucosylated antibodies with similar binding affinities that bound to regions spanning the GPC3 protein across unique epitopes. We found that for GPC3 targeting antibodies, the greatest activity in a Jurkat-NFAT reporter assay was observed for the afucosylated antibody that bound GPC3 most proximal to the membrane. Further, the antibody that bound membrane proximal also had the most potent activity in primary ACTR T cell cytotoxicity and cytokine release assays. The physical distance between T cells and tumor targets has been previously determined to impact T cell activation for both peptide-MHC and CAR-T interactions. Similarly, our results demonstrate a potential relationship between spatial distance of tumor targets and T cells in determining the activity ACTR transduced T cells when targeting GPC3. Our data demonstrate that ACTR T cell activity is antibody-specific and dose-titratable, highlighting both efficacy and improved safety of the ACTR T cell platform when targeting GPC3+ solid tumor malignancies. Citation Format: Greg Motz, John Shin, Kathleen Whiteman, Birgit Schultes, Tapasya Pai, Lori Westendorf, Seth Ettenberg, Travis Biechele, Django Sussman, Heather Huet. Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3762. doi:10.1158/1538-7445.AM2017-3762

Abstract 4605: Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody

B cell maturation antigen (BCMA) has recently emerged as an attractive therapeutic target in multiple myeloma. BCMA has restricted expression on plasma cells with little to no expression on other normal tissues, but is upregulated on the surface of multiple myeloma cells. BCMA can regulate proliferation and survival of myeloma cells via binding to its ligands APRIL and BAFF and induce downstream signaling pathways. Thus, several approaches to target BCMA are currently under clinical investigation, including chimeric antigen receptor (CAR) T cell therapies, bispecific antibodies and antibody drug conjugates. The Antibody-Coupled T cell Receptor (ACTR) technology is a universal, engineered T cell therapy consisting of the extracellular domain of human CD16 and the intracellular T cell co-stimulatory and signaling domains. ACTR is designed to engage the Fc domain of therapeutic antibodies opsonized to target cells to mediate anti-tumor activity. Previous work has demonstrated ACTR T cell activity in combination with rituximab, trastuzumab, and hu14.18 K322A against CD20, Her2, and GD2 expressing cell lines, respectively (Kudo et al. Cancer Res 2014; 74:93-103). Currently ACTR is being evaluated in Phase I clinical trials with rituximab to treat relapsed refractory B cell lymphoma. Here we demonstrate a humanized afucosylated anti-BCMA antibody, SEA-BCMA, binds to ACTR expressing T cells with high affinity and mediates T cell activation, potent cytotoxicity, cytokine release and proliferation across a wide range of BCMA expressing myeloma cells. ACTR activity was specific to SEA-BCMA - opsonized target cells, dose dependent and had no activity on BCMA negative tumor lines. Furthermore, the SEA-BCMA antibody has additional properties that might contribute to a therapeutic effect, including blocking the binding of ligands to BCMA and driving natural killer cell mediated ADCC effects. These preclinical studies demonstrate a promising multi-faceted activity of ACTR T cells in combination with the anti-BCMA antibody, SEA-BCMA, for clinical consideration in multiple myeloma patients. Citation Format: Tooba Cheema, Taylor Hickman, Katie O9Callaghan, Lori Westendorf, Luke Manlove, Shyra Gardai, Allison Nelson, Ryan Boomer, Kathleen McGinness, Birgit Schultes, Seth Ettenberg, Django Sussman, Heather Huet. Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2017-4605