Luc Desnoyers

Liver-specific Activities of FGF19 Require Klotho beta

Hepatocyte function is regulated by members of the fibroblast growth factor (FGF) family of proteins, but little is known about the specific molecular mechanisms of this endocrine pathway. FGF19 regulates bile acid homeostasis and gall bladder filling; FGF19 binds only to FGF receptor 4 (FGFR4), but its liver-specific activity cannot be explained solely by the distribution of this receptor. Although it has been suggested that Klotho beta (KLB) may have a role in mediating FGF19 activity, we have provided for the first time definitive evidence that KLB is required for FGF19 binding to FGFR4, intracellular signaling, and downstream modulation of gene expression. We have shown that FGFR4 is widely distributed in mouse, whereas KLB distribution is more restricted. Liver was the only organ in which both genes were abundantly expressed. We show that in mice, FGF19 injection triggers liver-specific induction of c-Fos and repression of CYP7A1. The tissue-specific activity of FGF19 supports the unique intersection of KLB and FGFR4 distribution in liver. These studies define KLB as a novel FGFR4 coreceptor required for FGF19 liver specific functions.

Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.

WISP-1 Is an Osteoblastic Regulator Expressed During Skeletal Development and Fracture Repair

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteoblastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.

Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.

A proteolytically activatable EGFR Probody demonstrates antitumor efficacy while alleviating toxicity. Seek and Destroy One of the main problems with current cancer therapies is lack of specificity: Traditional chemotherapeutics target all dividing cells, and even more restricted drugs, like monoclonal antibodies, may have on-target but off-tumor side effects. But what if you had a drug that was only turned on in the presence of the tumor? Desnoyers et al. now report the development of a Probody that targets epidermal growth factor receptor (EGFR) only in the presence of tumor. Cetuximab is a Food and Drug Administration–approved EGFR-targeting antibody used to treat metastatic colorectal cancer and head and neck cancer, but therapy often results in dose-limiting skin rash. The authors modified cetuximab to form a Probody (PB1)—where the antigen-binding sites are masked until the antibody is activated by proteases commonly found in the tumor microenvironment. The authors found that PB1 was largely inert while in circulation in mice, but that it had comparable efficacy to cetuximab in the presence of tumor. In nonhuman primates, PB1 demonstrated safety and decreased toxicity at higher doses than cetuximab. What’s more, ex vivo human primary tumor samples were sufficient to activate PB1. If these data hold true in human trials and for other antibodies, Probodies could be used to target cancer while minimizing treatment side effects. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)–directed Probody therapeutic—an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.

FGF19 and Cancer

Fibroblast growth factors (FGFs) and their cognate receptors, FGF receptors (FGFRs), play critical roles in a variety of normal developmental and physiological processes. Numerous reports support a role for deregulation of FGF-FGFR signaling, whether it is at the ligand and/or receptor level, in tumor development and progression. The FGF19-FGFR4 signaling axis has been implicated in the pathogenesis of several cancers, including hepatocellular carcinomas in mice and potentially in humans. This chapter focuses on recent progress in the understanding of the molecular mechanisms of FGF19 action and its potential involvement in cancer.

Abstract C158: Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic.

Probodies represent a unique class of antibody-based therapeutics that specifically target activity to diseased tissues including cancer. Probodies are designed to be inactive in circulation and in healthy tissue and activated only within the tumor microenvironment by dysregulated protease activity. The Probody approach enables promising therapeutic targets not druggable with conventional antibodies due to toxicity. Inhibition of Notch signaling has revealed the therapeutic potential of targeting this pathway in cancer; however, systemic inhibition results in severe toxicities (e.g. gastrointestinal and cardiac) in preclinical and clinical studies limiting the development of Notch-targeting therapeutics. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged1 and Jagged2 (Jagged1/2). In vitro cell based assays and in vivo ectopic xenograft mouse tumor models (BxPC3 and H292) show dose-dependent inhibition of Notch signaling and anti-tumor activity, respectively. However, the antibody also shows profound toxicities in mice, including weight loss, alopecia, hyperkeratosis, and athymia, due to on-target inhibition of Jagged1/2 in healthy tissue. To address these toxicities, we developed a fully recombinant anti-Jagged1/2 Probody, CTX-033, comprised of the same antibody but with a unique peptide mask that blocks the antigen binding site linked to the light chain of the antibody by a linker containing a substrate that is cleavable by one or more proteases upregulated in cancer. CTX-033 had similar anti-tumor activity in the mouse xenograft models as compared to the antibody. However, despite a two-fold higher systemic exposure to CTX-033 as compared to the antibody, Probody treated mice did not exhibit the toxicities associated with the antibody treatment. Furthermore, CTX-033 shows additive anti-tumor efficacy when combined with gemcitabine in the BxPC3 model. Notably, the Probody plus gemcitabine combination lacks the significant toxicity associated with the antibody plus gemcitabine combination treatment. To validate both Jagged expression and protease activity in patient tumor samples, we used a novel in situ assay, immunofluorescent Probody zymography. The results reveal broad expression of the Jagged ligands and specific activation and binding of CTX-033 in patient pancreatic adenocarcinoma tissue samples. The data described here with the anti-Jagged Probody, CTX-033, demonstrates (1) the potential of the Probody platform to enable the safe targeting of two key ligands in the Notch signaling pathway not possible with a traditional antibody format and (2) the presence of both Jagged1/2 and proteases capable of activating CTX-033 in patient tumor samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C158. Citation Format: Jason Sagert, James West, Olga Vasiljeva, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Annie Yang, Chihunt Wong, Elizabeth Menendez, Krishna Polu, Henry Lowman. Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C158.

Abstract 2665: Transforming Notch ligands into tumor-antigen targets: A Probody-Drug Conjugate (PDC) targeting Jagged 1 and Jagged 2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of antibody drug conjugates (ADC) holds significant promise for improving outcomes in patients with cancer. However, toxicity can limit the number of accessible targets for these highly potent and empowered antibody formats due to expression in healthy tissue. Probody formatted ADCs enable opening up the therapeutic window for high potential but previously inaccessible targets, such as the Jagged ligands in the Notch pathway. Probodies are fully recombinant biotherapeutics comprised of a monoclonal antibody whose binding to target antigen is blocked by a masking peptide. Upon cleavage of a specific substrate-linker by tumor-specific proteases, the activated Probody binds its target, resulting in tumor-localized activity. Jagged expression is observed in a wide variety of patient tumors including multiple myeloma, pancreatic cancer, breast cancer, and prostate cancer. We previously described a novel anti-Jagged 1/2 antibody that is efficacious in slowing tumor growth in mouse in-vivo tumor models but results in systemic toxicity. A corresponding Probody mitigates systemic toxicities associated with inhibition of Jagged-induced Notch signaling while maintaining anti-tumor efficacy. Here we show that the Notch ligands Jagged 1 and Jagged 2 have properties that could also enable an antibody-drug conjugate (ADC) approach because the ligands are both expressed on the cell surface and can internalize an anti-Jagged antibody. Using FACS we have shown that Jagged 1/2 are expressed on several human cancer cell lines and by fluorescent IHC staining, the expression of Jagged 1/2 is maintained in the corresponding xenograft tumors. To further explore the potential of Jagged as an ADC target, we engineered a Probody Drug Conjugate (PDC) conjugated to the microtubule inhibitor MMAE. This PDC is efficacious in a pancreatic xenograft tumor model BxPC3. Importantly, in the BxPC3 model the PDC shows equivalent in vivo efficacy to the corresponding ADC without causing the systemic toxicity associated with ADC treatment. Supportive of the potential clinical benefit of an anti-Jagged PDC, more than 75% of lung, pancreatic, and breast cancer patient tumor samples evaluated show moderate to high Jagged 1/2 expression as measured by IHC staining. These data demonstrate that the ProbodyTM platform has potential to enable the use of drug conjugates to target Jagged ligands in the Notch pathway. Citation Format: Jason Sagert, Jim West, Chihunt Wong, Luc Desnoyers, Olga Vasiljeva, Jennifer Richardson, Krishna Polu, Henry Lowman. Transforming Notch ligands into tumor-antigen targets: A Probody-Drug Conjugate (PDC) targeting Jagged 1 and Jagged 2. [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 2665. doi:10.1158/1538-7445.AM2014-2665

Abstract 4570: Development of a proteolytically activatable EGFR Probody for cancer therapy.

Antibodies directed to specific disease-related antigens have proven to be very successful therapeutics for a variety of disease indications. In spite of their high affinity and specificity for target antigen, target-mediated toxicity constitutes a major limitation for the development of antibodies to certain targets. We have addressed this type of on-target toxicity by developing a new class of targeting antibodies (Probody™ therapeutics) that remain in an inert, masked form until proteolytically activated at the site of disease. As a proof-of-concept for the construction of a Probody, we used cetuximab as a starting point. Cetuximab is an EGFR-targeted antibody approved for the treatment of colorectal and head-and-neck cancers that produces an on-target toxicity in the form of a skin rash that afflicts 88% of patients treated with the antibody. We engineered an EGFR Probody by incorporating an inhibitory masking peptide fused to the antibody light chain. Masking of the Probody is achieved through a linker that also incorporates a substrate that is cleaved by one or more proteases up-regulated in cancer. In vitro, EGFR binding and cell-based activities of the masked Probody were diminished compared to those of cetuximab, but treatment with exogenous target proteases activated the Probody and restored activity comparable to cetuximab. Using tumor xenograft models in mice, we demonstrated that the Probody remained masked in systemic circulation but was activated and accumulated in the tumor microenvironment. Tumor activation of the Probody translated to efficacy similar to that seen with cetuximab. Consistent with our results in mice, the Probody remained efficiently masked in non-human primates and did not cause skin toxicity such as that observed in animals treated with cetuximab. Together, these results demonstrate that antibody activity can be specifically targeted to diseased tissue by utilizing locally overexpressed proteases as activating agents, suggesting that a variety of antigens not previously amenable to an antibody therapeutic approach may be successfully addressed with Probodies. Citation Format: Luc R. Desnoyers, Annie Yang, Tony W. Liang, Stephen Moore, Jason Sagert, Daniel R. Hostetter, Elizabeth Menendez, Fei Han, Michael Krimm, Ken Wong, Jennifer Richardson, Jim W. West, Shouchun Liu, Olga Vasiljeva, Henry B. Lowman. Development of a proteolytically activatable EGFR Probody for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4570. doi:10.1158/1538-7445.AM2013-4570

Abstract 4717: Final results from the Phase I study of MetMAb, a monovalent antagonist antibody to the receptor Met, dosed as single agent and in combination with bevacizumab in patients with advanced solid malignancies

Background: The receptor tyrosine kinase Met and/or its ligand, hepatocyte growth factor (HGF), are frequently over-expressed in cancers. Aberrant Met activation can enhance invasion, proliferation, and survival and may promote angiogenesis. MetMAb was uniquely engineered as a recombinant, humanized, monovalent monoclonal antibody to act as an antagonist of HGF-induced Met signaling. Materials and Methods: This 3+3 dose-escalation study consisted of three phases: 1) dose-escalation evaluating 1, 4, 10, 15, 20 and 30 mg/kg IV Q3W; 2) expansion at 15mg/kg IV Q3W; and 3) combination testing MetMAb, at 10 and 15mg/kg IV Q3W, plus bevacizumab (15mg/kg Q3W). Pre- and post- dose serum was collected for evaluation of pharmacodynamic biomarkers that could be affected by inhibition of Met and/or VEGF signaling. Results: 43 patients were treated in this study (21 in escalation, 13 in expansion, and 9 in combination). MetMAb has a half-life of approximately 11 days, and there were no apparent PK interactions with bevacizumab. MetMAb was generally well tolerated, both alone and in combination. The most frequent treatment-related adverse events included: fatigue, peripheral edema and hypoalbuminemia. In patients treated with the combination, no Grade 3-5 treatment-related adverse events were reported; a Grade 1, and dose-limiting adverse event of hemoptysis was reported in a patient who had central-necrosis of pulmonary metastases. A patient with gastric carcinoma achieved a complete response after 4 cycles of single-agent MetMAb; this patient came off study after 10 cycles with a sustained complete response. Conclusions: MetMAb, when administered as a single-agent, or in combination with bevacizumab was generally safe and well tolerated. A Phase II trial testing MetMAb in combination with bevacizumab and paclitaxel in patients with triple negative breast cancer is currently ongoing, while a Phase III trial testing MetMAb in combination with erlotinib in advanced NSCLC patients is planned. 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 4717. doi:10.1158/1538-7445.AM2011-4717