Mario Filion

Mechanism and function of monoclonal antibodies targeting siglec-15 for therapeutic inhibition of osteoclastic bone resorption.

Background: Bone-resorbing osteoclasts express the Siglec-15 receptor on their plasma membrane. Results: Siglec-15 antibodies inhibit osteoclast differentiation and induce receptor endocytosis and degradation. Conclusion: These results establish that Siglec-15 antibodies target osteoclasts in vivo and reveal a likely mechanism of action. Significance: Siglec-15 could serve as a target of novel therapeutics for osteoporosis and cancer-associated bone loss. The use of monoclonal antibodies to target functionally important cell-surface proteins on bone-resorbing osteoclasts represents a promising approach for treatment of cancer-associated bone loss and other skeletal pathologies. Previously, we identified Siglec-15, a little studied sialic acid-binding receptor, as a candidate target that is highly up-regulated during osteoclast differentiation induced by the cytokine receptor activator of NF-κB ligand (RANKL). In this report, we confirm that Siglec-15 is localized to the plasma membrane where it can be targeted by monoclonal antibodies to inhibit differentiation of functional osteoclasts in vitro. Furthermore, we found that treatment of mice with these antibodies led to a marked increase in bone mineral density, consistent with inhibition of osteoclast activity. Interestingly, osteoblast numbers were maintained despite the anti-resorptive activity. At the molecular level, Siglec-15 interacts with the adapter protein DAP12 and can induce Akt activation when clustered on the osteoclast cell surface, which likely represents its normal signaling function. Importantly, we discovered that monoclonal antibodies induce rapid internalization, lysosomal targeting, and degradation of Siglec-15 by inducing receptor dimerization. This study defines a key regulatory node that controls osteoclast differentiation and activity downstream of RANKL and supports further development of Siglec-15 antibodies as a novel class of bone loss therapeutics.

Abstract 1467: AB-16B5, a therapeutic monoclonal antibody against human clusterin that blocks the epithelial-to-mesenchymal transition

Studies are increasingly implicating clusterin (CLU) as an important contributing factor in cancer promotion and invasion. Although there is evidence that secreted (s) CLU plays a pro-survival role in tumors, recently published results demonstrated that sCLU is also a potent stimulator of the epithelial-to-mesenchymal transition (EMT). A family of monoclonal antibodies (mAbs) specific for human sCLU was generated and a subset of these was found to inhibit the migration and invasion of several types of cancer cells. Importantly, this EMT-inhibiting subset of mAbs all bind to a specific amino acid sequence in sCLU and inhibit metastasis in vivo. The variable regions of the lead candidate mAb were modified using an in silico molecular modeling approach to generate a humanized IgG2, designated AB-16B5, which exhibited almost identical binding parameters compared to the original mouse antibody, with an apparent KD of 2 − 5 nM for recombinant human CLU. Treatment of 4T1 carcinoma cells with AB-16B5 inhibited TGFβ-induced EMT indicating that the biological activity in vitro was maintained in the humanized antibody. Moreover, AB-16B5 inhibited the motility of EMT6 cells in scratch assays and reduced the invasion of DU145 and PC-3 hormone-insensitive human prostate cancer cells when cultured in Matrigel. In animal studies, DU145 prostate cancer cells were implanted in SCID mice and treated with 5 mg/kg AB-16B5 twice per week as a monotherapy or in combination with the anti-mitotic drug, docetaxel (TxT). These experiments showed that the tumors in the AB-16B5 treated animals were 55% smaller than those in the control group. Furthermore, combining the AB-16B5 treatment with that of TxT caused a reduction of tumor size by 40% compared to the TxT-only group. PC-3-derived tumors in SCID mice treated with AB-16B5 showed a similar degree of tumor growth inhibition. Importantly, in both prostate cancer models, the mice exposed to AB-16B5 exhibited a marked increase in their overall survival. Furthermore, AB-16B5 treatment of Nude mice with intra-cardiac implantations of MDA-231 breast cancer cells resulted in a reduction of the number of metastatic bone lesions. The pharmacokinetic parameters of AB-16B5 are comparable to other IgG2 antibodies with its half-life being approximately 15 days following a bolus intravenous injection in mice. Finally, mice exposed to AB-16B5 at 10-times the therapeutic dose displayed no observable signs of toxicity and no major changes in serum biochemistry were detected. In conclusion, AB-16B5 is one of the rare therapeutic mAbs that directly targets EMT to reduce the invasion of tumors. Its therapeutic effects hold much promise to control metastasis from breast and prostate tumors, in addition to enhancing the response to chemotherapeutic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1467.

Abstract LB-33: Pre-clinical evaluation of AB-16B5, a monoclonal antibody specific for tumor-associated sCLU, demonstrates therapeutic potential as an inhibitor of EMT in prostate, pancreatic and lung cancer

Secreted clusterin (sCLU) exhibits elevated expression in several cancer indications. sCLU plays a pro-survival role in tumors and, more recently, it was found to be a potent stimulator of the epithelial-to-mesenchymal transition (EMT). These findings led to the development of a monoclonal antibody, AB-16B5, that interacts with a specific EMT-inducing domain in sCLU resulting in abrogation of migration and invasion of many types of cancer cells. In previous studies, AB-16B5 reduced the invasion of tumors in models of metastatic breast cancer suggesting that the antibody blocked EMT in vivo. Here we present pre-clinical findings in models of prostate cancer, pancreatic cancer, and NSCLC. DU145 and PC-3 prostate cancer cells implanted in SCID mice grew slower in the groups treated with AB-16B5 as a monotherapy or in combination with docetaxel. This observation suggested that blocking sCLU with AB-16B5 enhanced the chemo-responsiveness to cytotoxic drugs, a finding that was consistent with the proposed role of sCLU as an inhibitor of apoptosis in cancer cells. Immunohistochemical examination of sections prepared from the prostate tumors exposed to AB-16B5 showed an increase in E-cadherin, a marker of epithelial cells, and a reduction of vimentin, a marker of mesenchymal cells, which indicated that EMT was blocked or even reversed in vivo. Additional analyses of tumors derived from human pancreatic cancer showed that sCLU was expressed in this indication as well, especially in tumors that were resistant to gemcitabine. In agreement with the role of sCLU as an inducer of EMT, AB-16B5 inhibited the invasion of PANC-1 pancreatic cancer cells in vitro. Importantly, treatment of SCID mice harboring BxPC-3 pancreatic tumors with AB-16B5 resulted in a significant enhancement of the response to gemcitabine. Similarly, the growth of tumors grown from the NSCLC cell line, A549, was also inhibited by AB-16B5. To explore the behavior of AB-16B5 in cynomolgus monkeys, the antibody was administered by i.v. infusion every 14 days at two doses of 10 and 50 mg/kg. Results indicated that AB-16B5 was well tolerated and no signs of toxicity were observed. Finally, to address the mechanism of action of sCLU, cell biology experiments indicated that sCLU is internalized in cancer cells, which leads to the activation of critical signaling pathways that favor cell proliferation and survival, including those pathways leading to a stimulation of NF-kappaB. Our studies imply that the combination of increased epithelial character of tumor cells exposed to AB-16B5 coupled with a reduction in the activation of survival pathways contribute to an increase in the sensitivity to chemotherapy and a significant reduction of tumor growth, in particular in cancer types such as pancreatic cancer, where EMT likely contributes to increased chemo-resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-33. doi:10.1158/1538-7445.AM2011-LB-33

Abstract LB-091: Inhibition of Siglec-15 prevents bone loss in a mouse model of multiple myeloma

Multiple myeloma (MM) is a B-cell malignancy associated with infiltration and growth of plasma cells in the bone marrow resulting in end-organ damage including devastating osteolytic bone disease. We examined the ability of 25B2, an antibody targeting Siglec-15, to prevent bone loss in the 5T2MM syngeneic model of MM. C57BlKaLwRij male mice (n = 10/group) were injected with 5T2MM cells and 24h later treated for 12 weeks with the PBS, 25B2 (10 mg/kg, q3d), bortezomib (0.7 mg/kg, q3d) or a combination of 25B2 and bortezomib. 25B2 treatment caused a 3.7-, 5.4- and 1.2-fold increase in bone volume (%BV/TV) compared to PBS-treated mice in the tibia, femur and lumbar vertebra (LV), respectively. Increases in bone volume were also seen with bortezomib with 3.1-, 4.1- and 1.4-fold increases in the tibia, femur and LV, respectively. Combination of the two agents further increased%BV/TV by 6.9-, 10.5- and 1.5-fold in the tibia, femur and LV, respectively, when compared to the PBS treated mice. All of the changes were statistically significant with the exception of those in the LV. Unlike bortezomib treatment, which inhibits the growth of 5T2MM cells in this model, there was no direct effect of 25B2 on tumor burden in 5T2MM mice when the percentage of plasmacytosis and the serum paraprotein levels were measured. However, 25B2 caused a significant decrease in the number of bone lesions by 49.5%. The number of lesions in the bortezomib group decreased by 71.7%, but there was no significant difference between the 25B2 and bortezomib groups. The number of lesions decreased by 89.4% in the combination group. Consistent with the mechanism of action of Siglec-15 antibodies, osteoclasts were rendered inactive, however cross-talk with bone-forming osteoblasts was likely maintained. This unique mechanism of action would be particularly appropriate for MM, where osteolytic disease is not only the result of increased osteoclast activity but also decreased osteoblast-mediated bone formation. The 5T2MM model is one of the most representative of the human pathology and therefore, the effect observed in response to the Siglec-15 antibody holds the promise that such a treatment in MM patients could provide benefit and further delay skeletal related events. Citation Format: Gilles B. Tremblay, Anna Moraitis, Karin Vanderkerken, Mario Filion. Inhibition of Siglec-15 prevents bone loss in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-091.

Abstract 666: AB-3A4: A novel KAAG1-targeting antibody-drug conjugate is active in models of ovarian carcinoma, triple-negative breast cancer and castration-resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Starting from total RNA prepared from late-stage serous ovarian tumors and low-malignancy potential tumors, a sensitive subtractive cloning technology called STAR was used to identify differentially expressed sequences that were up-regulated in the malignant tumors. This analysis led to the identification of KAAG1, an antigen that is specifically expressed in the vast majority of the ovarian tumors. Monoclonal antibodies (mAbs) that bind to human KAAG1 were generated and one of these, AB-3A4, interacted with KAAG1 expressed on the surface of several ovarian cancer, triple-negative breast cancer (TNBC) and prostate cancer cell lines. Further studies indicated that the AB-3A4/KAAG1 complex was rapidly internalized and co-localized with LAMP1, a marker of lysosomes, indicating that AB-3A4 has potential as an antibody-drug conjugate (ADC). Here we describe preclinical results of AB-3A4 conjugated to the cytotoxic agent monomethyl auristatin E (AB-3A4-vcMMAE). Binding studies with KAAG1-positive cell lines, such as the SKOV-3 (ovarian), MDA-MB-231 (TNBC) and PC-3 (prostate) showed that AB-3A4-vcMMAE interacted with its antigen in a similar manner to the unconjugated AB-3A4. In cytotoxicity assays, AB-3A4-vcMMAE killed cancer cell lines expressing KAAG1 with an IC50 value of 1 - 2 nM, whereas it displayed no specific potency in KAAG1-low or -negative cells. SKOV-3 cells were subcutaneously implanted in SCID mice and allowed to grow to a volume of 150 - 200 mm3 after which four injections of AB-3A4-vcMMAE at 5 mg/kg were administered over two weeks. Rapid and sustained tumor regression was observed for several weeks post-injection. In PC-3 xenografts, a single injection of AB-3A4-vcMMAE at 3 mg/kg was sufficient to cause significant tumor regression and this efficacy was found to be dose-dependent between doses of 1 and 10 mg/kg. In parallel treatment groups, no significant tumor shrinkage was observed with either a control IgG-vcMMAE or a mixture of unconjugated AB-3A4 and free MMAE at a drug-antibody ratio corresponding to that of AB-3A4-vcMMAE. Furthermore, xenografts of TNBC MDA-MB-231 cells were significantly regressed following a single injection of AB-3A4-vcMMAE with no recurrence after several weeks post-treatment. Finally, using an intraperitoneal orthotopic model of ovarian cancer, treatment with AB-3A4-vcMMAE significantly increased the overall survival of mice compared to mice treated with the control ADC. Collectively, our results demonstrate the potent efficacy of AB-3A4-vcMMAE in several disease models and underscore the potential of this novel agent in ovarian cancer, TNBC and castration-resistant prostate cancer, three areas for which very few targeted therapies exist. Citation Format: Gilles B. Tremblay, Anna Moraitis, Dominique Bedard, Adriana Orimoto, Martine Page, Anne-Marie Mes-Masson, Mario Filion. AB-3A4: A novel KAAG1-targeting antibody-drug conjugate is active in models of ovarian carcinoma, triple-negative breast cancer and castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2014-666