Development of Anti-CD32b Antibodies with Enhanced Fc Function for the Treatment of B and Plasma Cell Malignancies.

The sole inhibitory Fcγ receptor CD32b (FcγRIIb) is expressed throughout B and plasma cell development and on their malignant counterparts. CD32b expression on malignant B cells is known to provide a mechanism of resistance to rituximab that can be ameliorated with a CD32b-blocking antibody. CD32b, therefore, represents an attractive tumor antigen for targeting with a monoclonal antibody (mAb). To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. Their complementarity-determining regions (CDR) bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated to enhance activation of FcγRIIIa on immune effector cells. The NVS32b mAbs selectively target CD32b+ malignant cells and healthy B cells but not myeloid cells. They mediate potent killing of opsonized CD32b+ cells via antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) as well as complement-dependent cytotoxicity (CDC). In addition, NVS32b CDRs block the CD32b Fc-binding domain, thereby minimizing CD32b-mediated resistance to therapeutic mAbs including rituximab, obinutuzumab, and daratumumab. NVS32b mAbs demonstrate robust antitumor activity against CD32b+ xenografts in vivo and immunomodulatory activity including recruitment of macrophages to the tumor and enhancement of dendritic cell maturation in response to immune complexes. Finally, the activity of NVS32b mAbs on CD32b+ primary malignant B and plasma cells was confirmed using samples from patients with B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. The findings indicate the promising potential of NVS32b mAbs as a single agent or in combination with other mAb therapeutics for patients with CD32b+ malignant cells.

Cyclophosphamide Enhances Cancer Antibody Immunotherapy in the Resistant Bone Marrow Niche by Modulating Macrophage FcγR Expression.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.

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.

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.

Differential expression of RON in small and non-small cell lung cancers.

RON is a MET related receptor tyrosine kinase (RTK) and its natural ligand is macrophage stimulating protein (MSP). RON plays a very important role in the regulation of inflammation. Several studies have previously reported overexpression of RON in a variety of cancers including lung and identified numerous RON alternate splice forms that very likely contribute to tumor growth and metastasis. Here, we have analyzed the expression of total RON protein as well as its kinase-active form (phospho-RON) in 175 archival lung tumor FFPE (formalin fixed paraffin embedded) samples that included non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and their metastatic forms. The frequency and intensity of RON protein expression was much higher in lung tumors of neuroendocrine origin such as SCLC and in secondary tumors that metastasized to brain. In addition, the majority of the expressed RON protein was phospho-RON. We also identified 62, and 30 kDa isoforms of RON (GenBank accession numbers are JN689381 and JN689382) using RNA isolated from pooled lung cancer cell lines and RT-PCR. A majority of the NSCLC cell lines expressed a 150 kDa band that corresponded to the RON ß chain and 120 kDa band in the panel of SCLC cell lines tested. RON was expressed on the cell surface in NSCLC cell lines. Finally, knock down of RON expression resulted in a significant loss in viability as well as motility in lung cancer cells suggesting that RON is a potential therapeutic target.

RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma.

RON (MST1R) is one of two members of the MET receptor tyrosine kinase family, along with parent receptor MET. RON has a putative role in several cancers, but its expression and function is poorly characterized in gastroesophageal adenocarcinoma. A recognized functional role of MET tyrosine kinase in gastroesophageal cancer has led to early phase clinical trials using MET inhibitors, with unimpressive results. Therefore, the role of RON in gastroesophageal cancer, as well as its role in cooperative signaling with MET and as a mechanism of resistance to MET inhibition, was studied in gastroesophageal tissues and cell lines. By IHC, RON was highly over-expressed in 74% of gastroesophageal samples (n=94), and over-expression was prognostic of poor survival (p=0.008); RON and MET co-expression occurred in 43% of samples and was prognostic of worst survival (p=0.03). High MST1R gene copy number by quantitative polymerase chain reaction, and confirmed by fluorescence in situ hybridization and/or array comparative genomic hybridization, was seen in 35.5% (16/45) of cases. High MST1R gene copy number correlated with poor survival (p=0.01), and was associated with high MET and ERBB2 gene copy number. A novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON signaling was functional in cell lines, activating downstream effector STAT3, and resulted in increased viability over controls. RON and MET co-stimulation assays led to enhanced malignant phenotypes over stimulation of either receptor alone. Growth inhibition as evidenced by viability and apoptosis assays was optimal using novel blocking monoclonal antibodies to both RON and MET, versus either alone. SU11274, a classic MET small molecule tyrosine kinase inhibitor, blocked signaling of both receptors, and proved synergistic when combined with STAT3 inhibition (combination index < 1). These preclinical studies define RON as an important novel prognostic marker and therapeutic target for gastroesophageal cancer warranting further investigation.

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.

Functional characterization of integrin alpha6beta4 adhesion interactions using soluble integrin constructs reveals the involvement of different functional domains in the beta4 subunit.

Integrin alpha6beta4-mediated adhesion interactions play key roles in keratinocyte and epithelial tumor cell biology. In order to evaluate how alpha6beta4 adhesion interactions contribute to these important cellular processes, the authors generated soluble versions of the integrin by recombinant expression of the subunit ectodomains fused to a human immunoglobulin G (IgG) Fc constant domain. Coexpression of the appropriate subunits enabled dimerization, secretion and purification of stable Fc-containing alpha6beta4 heterodimers. The soluble proteins exhibited the same metal ion and ligand dependency in their binding characteristics as intact alpha6beta4. Using these reagents in combination with anti-beta4 antibodies, the authors identified two distinct functional epitopes on the beta4 subunit. They demonstrated the involvement of one epitope in adhesion interactions and the other in regulating adhesion-independent growth in alpha6beta4-expressing tumor cell lines. The availability of these soluble integrin reagents and the data provided herein help to further delineate the structure-function relationships regulating alpha6beta4 signaling biology.