David Stover

Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma

Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)-hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated beta-catenin level while down-regulating nuclear factor-kappaB (NF-kappaB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

Inhibiting Dickkopf-1 (Dkk1) removes suppression of bone formation and prevents the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf‐1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti‐Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti‐Dkk1 treatment prevented 5T2MM‐induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. μCT analysis showed that anti‐Dkk1 treatment significantly protected against 5T2MM‐induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.

Serum concentrations of DKK‐1 correlate with the extent of bone disease in patients with multiple myeloma

Objectives:  Lytic bone disease is a hallmark of multiple myeloma (MM) and is caused by osteoclast activation and osteoblast inhibition. Secretion of Dickkopf (DKK)‐1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. So far, there is no study showing a significant difference in serum DKK‐1 levels in MM patients with or without lytic bone lesions.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Modulation of the SH2 Binding Specificity and Kinase Activity of Src by Tyrosine Phosphorylation within Its SH2 Domain

The Src family of kinases are held in an inactive state by interaction of their SH2 domain with a C-terminal phosphotyrosine. Dephosphorylation of this site can reactivate Src; however, recent evidence suggests that activation can also occur without dephosphorylation. In this study, platelet-derived growth factor receptor phosphorylation of Src on Tyr-213 specifically blocked binding of its SH2 domain to a phosphopeptide corresponding to the C-terminal regulatory sequence, while binding to other sequences, such as the platelet-derived growth factor receptor or a peptide from the epidermal growth factor receptor, was unaffected. Consequently, Src was activated over 50-fold. This is the first demonstration of regulation of a SH2 domain specificity by post-translational modification and is likely to be a general mechanism for regulation of all Src-like kinases.

In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

Serum concentrations of DKK-1 decrease in patients with multiple myeloma responding to anti-myeloma treatment.

Lytic bone destruction is a hallmark of multiple myeloma (MM) and is because of an uncoupling of bone remodeling. Secretion of Dickkopf (DKK)‐1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. In this study, the effect of different treatment regimens for MM on serum DKK‐1 was evaluated and correlated with the response to treatment in 101 myeloma patients receiving bortezomib, thalidomide, lenalidomide, adriamycin and dexamethasone (AD) or high‐dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT). At baseline, myeloma patients had increased serum DKK‐1 as compared with patients with MGUS (mean 3786 pg/mL vs. 1993 pg/mL). There was no difference between previously untreated MM patients and patients at relapse. A significant decrease of DKK‐1 after therapy was seen in the following groups: Bortezomib (4059 pg/mL vs. 1862 pg/mL, P = 0.016), lenalidomide (11837 pg/mL vs. 4374 pg/mL, P = 0.039), AD (1668 pg/mL vs. 1241 pg/mL, P = 0.016), and AD + HDCT + ASCT (2446 pg/mL vs. 1082 pg/mL, P = 0.001). Thalidomide led to a non‐significant decrease in DKK‐1 (1705 pg/mL vs. 1269 pg/mL, P = 0.081). Within all groups, a significant decrease of DKK‐1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non‐responders. We show for the first time that serum DKK‐1 levels decrease in myeloma patients responding to treatment, irrespective of the regimen chosen. These data suggest that myeloma cells are the main source of circulating DKK‐1 protein and provide a framework for clinical trials on anti‐DKK‐1 treatment in MM.

TRAIL signals to apoptosis in chronic lymphocytic leukaemia cells primarily through TRAIL-R1 whereas cross-linked agonistic TRAIL-R2 antibodies facilitate signalling via TRAIL-R2

Tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL), a member of the TNF family, which is being developed as an anti‐tumour agent due to its selective toxicity to tumour cells, induces apoptosis by binding to two membrane‐bound receptors, TRAIL‐R1 and TRAIL‐R2. Clinical trials have been initiated with various preparations of TRAIL as well as agonistic monoclonal antibodies to TRAIL‐R1 and TRAIL‐R2. Previously we reported that prior treatment of primary chronic lymphocytic leukaemia (CLL) cells with histone deacetylase inhibitors was required to sensitize CLL cells to TRAIL and, using various receptor‐selective TRAIL mutant ligands, we demonstrated that CLL cells signalled to apoptosis primarily through TRAIL‐R1. Some, but not all, agonistic TRAIL‐receptor antibodies require cross‐linking in order to induce apoptosis. The present study demonstrated that CLL cells can signal to apoptosis through the TRAIL‐R2 receptor, but only after cross‐linking of the agonistic TRAIL‐R2 antibodies, LBY135 and lexatumumab (HGS‐ETR2). In contrast, signalling through TRAIL‐R1 by receptor‐selective ligands or certain agonistic antibodies, such as mapatumumab (HGS‐ETR1), occurs in the absence of cross‐linking. These results further highlight important differences in apoptotic signalling triggered through TRAIL‐R1 and TRAIL‐R2 in primary tumour cells. Such information is clearly important for the rational optimisation of TRAIL therapy in primary lymphoid malignancies, such as CLL.

A potent protein-tyrosine kinase inhibitor which selectively blocks proliferation of epidermal growth factor receptor-expressing tumor cells in vitro and in vivo.

A calculated 3‐D model of the kinase domain of the epidermal growth factor receptor (EGF‐R) protein‐tyrosine kinase (PTK) was used to develop a pharmacophore model for ATP‐competitive inhibitors and, subsequently, a new class of selective EGF‐R kinase inhibitors. CGP 59326A, a highly selective and potent inhibitor of the EGF‐R in vitro, inhibited the proliferation of EGF‐R‐expressing epithelial lines, while having little anti‐proliferative activity against EGF‐R‐negative lines. In contrast to previously described inhibitors, CGP 59326A had potent and selective in vivo anti‐tumor activity at well‐tolerated doses against EGF‐R‐expressing tumors (e.g., ED50 of 0.78 to 1.5 mg/kg for inhibition of A431 tumor growth). CGP 59326A inhibited growth of human tumor xenografts expressing the EGF‐R but showed little activity against EGF‐R‐negative xenografts. Combination of CGP 59326A with cytotoxic agents resulted in tumor regression and cures. The high selectivity and attractive biological profile of CGP 59326A suggest that it could have therapeutic value in the treatment of proliferative diseases which involve mitogenic signaling from the EGF‐R. Int. J. Cancer 76:154–163, 1998.© 1998 Wiley‐Liss, Inc.

Differential phosphoprofiles of EGF and EGFR kinase inhibitor-treated human tumor cells and mouse xenografts

The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 106 cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.