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.

Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody-Drug Conjugate.

Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.

Model-Informed Drug Development of the Masked Anti-PD-L1 Antibody CX-072.

CX-072 is an anti-PD-L1 (programmed death ligand 1) Probody therapeutic (Pb-Tx) designed to be preferentially activated by proteases in the tumor microenvironment and not in healthy tissue. Here, we report the model-informed drug development of CX-072. A quantitative systems pharmacology (QSP) model that captured known mechanisms of Pb-Tx activation, biodistribution, elimination, and target engagement was used to inform clinical translation. The QSP model predicted that a trough level of masked CX-072 (intact CX-072) of 13-99 nM would correspond to a targeted, 95% receptor occupancy in the tumor. The QSP model predictions appeared consistent with preliminary human single-dose pharmacokinetic (PK) data following CX-072 0.03-30.0 mg/kg as monotherapy: CX-072 circulated predominantly as intact CX-072 with minimal evidence of target-mediated drug disposition. A preliminary population PK (POPPK) analysis based upon 130 subjects receiving 0.03-30.0 mg/kg as monotherapy included a provision for a putative time-dependent and dose-dependent antidrug antibody (ADA) effect on clearance (CL) with a mixture model. Preliminary POPPK estimates for intact CX-072 time-invariant CL and volume of distribution were 0.306 L/day and 4.84 L, respectively. Exposure-response analyses did not identify statistically significant relationships with best change from baseline sum of measurements and either adverse events of grade ≥ 3 or of special interest. Simulations suggested that > 95% of patients receiving CX-072 10 mg/kg every two weeks would exceed the targeted trough level regardless of ADA, and that dose adjustment by body weight was not necessary, supporting a fixed 800 mg dose for evaluation in phase II.

Structural basis and therapeutic implication of the interaction of CCN proteins with glycoconjugates.

The CCN family of growth factors is composed of six structurally related proteins including the cysteine-rich 61 (Cyr61), connective tissue growth factor (CTGF), nephroblastoma overexpressed (NOV), Wnt-1 induced secreted protein-1 (WISP-1), WISP-2 and WISP-3. Each family member consists of four conserved cysteine rich modular domains with sequence similarity to the insulin like growth factor binding proteins, von Willebrand factor, thrombospondin repeat and the growth factors cysteine knot. The CCN proteins demonstrate a wide variety of biological activities regulating cell adhesion, proliferation, survival, migration, invasion in vitro and tumorigenesis and angiogenesis in vivo. Both cancer promoting and inhibiting roles were proposed for several CCN proteins suggesting that contextual factors could regulate their activities. Consistent with this hypothesis, structural and experimental evidence indicate that the function of these proteins is modulated by their interaction with sulfated glycosaminoglycans. Because the CCN proteins are implicated in the tumorigenic process, they are potential targets for the development of cancer therapeutics. Modulation of their glycosaminoglycan interaction by exogenous, highly sulfated polysaccharides, oligosaccharides or glycosaminoglycan mimetics could prevent their participation in cancer progression. Understanding the structural requirements for their polysaccharide interaction should provide important information to generate glycosaminoglycan-based cancer therapeutics targeting the CCN family of proteins.

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

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.

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.

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.

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.

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.