Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma.

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

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.

Unexpected hepatotoxicity in a phase I study of TAS266, a novel tetravalent agonistic Nanobody® targeting the DR5 receptor.

PURPOSE: TAS266 is a novel agonistic tetravalent Nanobody(®) targeting the DR5 receptor. In preclinical studies, TAS266 was more potent than a cross-linked DR5 antibody or TRAIL. This first-in-human study was designed to evaluate the safety and tolerability, maximum tolerated dose, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary efficacy of TAS266. METHODS: Adult patients with advanced solid tumors were to receive assigned doses of TAS266 (3, 10, 15, or 20 mg/kg) intravenously on days 1, 8, 15, and 22 of a 28-day treatment cycle. RESULTS: Grade ≥3 elevations in aspartate aminotransferase and/or alanine aminotransferase levels, occurring during cycle 1 in three of four patients at the 3 mg/kg dose level, were attributed to TAS266 and led to early study termination. Liver enzyme levels quickly returned to grade ≤1 following TAS266 discontinuation. Evidence of preexisting antibodies able to bind to TAS266 was found in the three patients who experienced these dose-limiting toxicities. Immunogenic responses remained elevated and strengthened at end-of-treatment (EOT). In the one patient who did not develop hepatotoxicity, no evidence of immunogenicity was observed at baseline or following administration of 4 TAS266 doses; however, incipient positive immunogenicity was observed at the EOT visit. CONCLUSION: TAS266 was associated with unexpected, significant but reversible hepatotoxicity. Although the underlying mechanism is not fully elucidated, factors including the molecule's high potency, immunogenicity to TAS266, and possibly increased DR5 expression on hepatocytes further enhancing the activity of the Nanobody(®) may have contributed to enhanced DR5 clustering and activation of hepatocyte apoptosis.

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.

Integrin-blocking antibodies delay keratinocyte re-epithelialization in a human three-dimensional wound healing model.

The alpha6beta4 integrin plays a significant role in tumor growth, angiogenesis and metastasis through modulation of growth factor signaling, and is a potentially important therapeutic target. However, alpha6beta4-mediated cell-matrix adhesion is critical in normal keratinocyte attachment, signaling and anchorage to the basement membrane through its interaction with laminin-5, raising potential risks for targeted therapy. Bioengineered Human Skin Equivalent (HSE), which have been shown to mimic their normal and wounded counterparts, have been used here to investigate the consequences of targeting beta4 to establish toxic effects on normal tissue homeostasis and epithelial wound repair. We tested two antibodies directed to different beta4 epitopes, one adhesion-blocking (ASC-8) and one non-adhesion blocking (ASC-3), and determined that these antibodies were appropriately localized to the basal surface of keratinocytes at the basement membrane interface where beta4 is expressed. While normal tissue architecture was not altered, ASC-8 induced a sub-basal split at the basement membrane in non-wounded tissue. In addition, wound closure was significantly inhibited by ASC-8, but not by ASC-3, as the epithelial tongue only covered 40 percent of the wound area at 120 hours post-wounding. These results demonstrate beta4 adhesion-blocking antibodies may have adverse effects on normal tissue, whereas antibodies directed to other epitopes may provide safer alternatives for therapy. Taken together, we conclude that these three-dimensional tissue models provide a biologically relevant platform to identify toxic effects induced by candidate therapeutics, which will allow generation of findings that are more predictive of in vivo responses early in the drug development process.

Antibodies directed to alpha6beta4 highlight the adhesive and signaling functions of the integrin in breast cancer cell lines.

Integrin alpha6beta4 signaling interactions have been implicated in tumor progression, and beta4 expression has been linked to poor prognosis in certain breast cancer subtypes. We generated human antibodies to alpha6beta4 to further evaluate its role in tumor cell signaling. Biochemical characterization indicated these antibodies are specific for alpha6beta4, recognize distinct epitopes and have low nanomolar affinities for both human and murine protein. The antibodies demonstrated differing effects on alpha6beta4-mediated cellular adhesion, highlighting the existence of different functional epitopes on alpha6beta4. Interestingly however both antibodies blocked adhesion-independent growth in a panel of breast cancer cell lines. Antibody induced apoptosis and inhibition of phosphoinositide 3-kinase (PI3K) signaling were also observed within the context of matrix adhesion. Enhanced inhibitory effects were observed when the alpha6beta4 antibodies were used in combination with antibodies to epidermal growth factor receptor (EGFR) or erythoblastic leukemia viral oncogene homolog 2 (ErbB2). These findings illustrate a role for both the adhesive and signaling functions of alpha6beta4 in breast cancer cell survival. The antibodies and data generated herein advance our understanding of alpha6beta4 in regulating tumorigenic processes, and suggest that combination therapies involving alpha6beta4 may be therapeutically effective in breast cancer.