Development of a Biosimilar of Agalsidase Beta for the Treatment of Fabry Disease: Preclinical Evaluation.

BACKGROUND AND OBJECTIVE: Fabry disease (FD) is a rare lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A (aGal A). Since 2001, two different enzyme replacement therapies have been authorized, with agalsidase beta being used in most parts of the Western world. Currently, biosimilars of several expensive enzyme therapies are under development to improve their accessibility for patients. We present the preclinical results of the development of a biosimilar to agalsidase beta. METHODS: Produced in a Chinese hamster ovary (CHO)-cell system, the biosimilar aGal A Biosidus (AGABIO), was compared with agalsidase beta with respect to amino acid sequence, glycosylation, specific α-galactosidase activity, stability in plasma, and effects on cultured human Fabry fibroblasts and Fabry mice. RESULTS: AGABIO had the same amino acid composition and similar glycosylation, enzymatic activity, and stability as compared with agalsidase beta. After uptake in fibroblasts, α-galactosidase A activity increased in a dose-dependent manner, with maximum uptake observed after 24 h, which remained stable until at least 48 h. Both enzymes were localized to lysosomes. Reduction of accumulated globotriaosylceramide (Gb3) and lysoGb3 in cultured Fabry fibroblasts by AGABIO and agalsidase beta showed comparable dose-response curves. In Fabry knockout mice, after a single injection, both enzymes were rapidly cleared from the plasma and showed equal reductions in tissue and plasma sphingolipids. Repeated dose studies in rats did not raise any safety concerns. Anti-drug antibodies from patients with FD treated with agalsidase beta showed equal neutralization activity toward AGABIO. CONCLUSION: These findings support the biosimilarity of AGABIO in comparison with agalsidase beta. The clinical study phase is currently under development.

Combining inhibition of galectin-3 with and before a therapeutic vaccination is critical for the prostate-tumor-free outcome.

BACKGROUND: Prostate cancer (PCa) is a major health problem worldwide. Taxol derivatives-based chemotherapies or immunotherapies are usually proposed depending on the symptomatic status of the patient. In the case of immunotherapy, tumors develop robust immune escape mechanisms that abolish any protective response, and to date why prostate cancer is one of the most resistant diseases remains unresolved. METHODS: By using a combination of clinical data to study the transcriptome of metastasis samples from patients with castration-refractory prostate cancer, and state of the art cellular and molecular biology assays in samples from tumor-bearing mice that have been submitted to surgical resection of the tumor before receiving a vaccination, we answered several essential questions in the field of immunotherapy for prostate cancer. We also used two different methods to inhibit the expression of galectin-3 (Gal-3) in tumor cells: a stable RNA interference method to control the expression of this galectin efficiently only in tumor cells, and low and non-cytotoxic doses of docetaxel to easily transfer our findings to clinical settings. RESULTS: Herein, we show for the first time that Gal-3 expressed by prostate tumor cells is the main immune checkpoint responsible for the failure of vaccine-based immunotherapy. Our results show that low and non-cytotoxic doses of docetaxel lead to the inhibition of Gal-3 expression in PCa cells as well as in clinical samples of patients with metastatic and castration-resistant PCa promoting a Th1 response. We thus optimized a prostate cancer animal model that undergoes surgical resection of the tumor to mimic prostatectomy usually performed in patients. Importantly, using Gal-3-knocked down-PCa cells or low and non-cytotoxic doses of taxane before vaccination, we were able to highly control tumor recurrence through a direct impact on the proliferation and infiltration of CD8+ cytotoxic T. CONCLUSIONS: Thus, Gal-3 expression by PCa cells is a crucial inhibitor for the success of immunotherapy, and low doses of docetaxel with non-cytotoxic effect on leukocyte survival could be used before immunotherapy for all patients with PCa to reduce the expression of this critical negative immune checkpoint, pre-conditioning the tumor-microenvironment to activate an antitumor immune response and promote tumor-free outcome.

Stable and high expression of Galectin-8 tightly controls metastatic progression of prostate cancer.

Two decades ago, Galectin-8 was described as a prostate carcinoma biomarker since it is only expressed in the neoplastic prostate, but not in the healthy tissue. To date, no biological function has been attributed to Galectin-8 that could explain this differential expression. In this study we silenced Galectin-8 in two human prostate cancer cell lines, PC3 and IGR-CaP1, and designed a pre-clinical experimental model that allows monitoring the pathology from its early steps to the long-term metastatic stages. We show for the first time that the natural and conserved expression of Gal-8 in tumour cells is responsible for the metastatic evolution of prostate cancer. In fact, Gal-8 controls the rearrangement of the cytoskeleton and E-Cadherin expression, with a major impact on anoikis and homotypic aggregation of tumour cells, both being essential processes for the survival of circulating tumour cells during metastasis. While localized prostate cancer can be cured, metastatic and advanced disease remains a significant therapeutic challenge, urging for the identification of prognostic markers of the metastatic process. Collectively, our results highlight Galectin-8 as a potential target for anti-metastatic therapy against prostate cancer.

In Vivo Hemin Conditioning Targets the Vascular and Immunologic Compartments and Restrains Prostate Tumor Development.

Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model.Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes.Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1-conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels.Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response. Clin Cancer Res; 23(17); 5135-48. ©2017 AACR.

Galectin-1 triggers epithelial-mesenchymal transition in human hepatocellular carcinoma cells.

Galectin-1 (Gal1), a ß-galactoside-binding protein abundantly expressed in tumor microenvironments, is associated with the development of metastasis in hepatocellular carcinomas (HCC). However, the precise roles of Gal1 in HCC cell invasiveness and dissemination are uncertain. Here, we investigated whether Gal1 mediate epithelial-mesenchymal transition (EMT) in HCC cells, a key process during cancer progression. We used the well-differentiated and low invasive HepG2 cells and performed 'gain-of-function' and 'loss-function' experiments by transfecting cells with Gal1 cDNA constructs or by siRNA strategies, respectively. Epithelial and mesenchymal markers expression, changes in apico-basal polarity, independent-anchorage growth, and activation of specific signaling pathways were studied using Western blot, fluorescence microscopy, soft-agar assays, and FOP/TOP flash reporter system. Gal1 up-regulation in HepG2 cells induced down-regulation of the adherens junction protein E-cadherin and increased expression of the transcription factor Snail, one of the main inducers of EMT in HCC. Enhanced Gal1 expression facilitated the transition from epithelial cell morphology towards a fibroblastoid phenotype and favored up-regulation of the mesenchymal marker vimentin in HCC cells. Cells overexpressing Gal1 showed enhanced anchorage-independent growth and loss of apico-basal polarity. Remarkably, Gal1 promoted Akt activation, ß-catenin nuclear translocation, TCF4/LEF1 transcriptional activity and increased cyclin D1 and c-Myc expression, suggesting activation of the Wnt pathway. Furthermore, Gal1 overexpression induced E-cadherin downregulation through a PI3K/Akt-dependent mechanism. Our results provide the first evidence of a role of Gal1 as an inducer of EMT in HCC cells, with critical implications in HCC metastasis.

Study of galectins in tumor immunity: strategies and methods.

During the past decade, a better understanding of the cellular and molecular mechanisms underlying tumor immunity has provided the appropriate framework for the development of therapeutic strategies for cancer immunotherapy. Under this complex scenario, galectins have emerged as promising molecular targets for cancer therapy responsible of creating immunosuppressive microenvironments at sites of tumor growth and metastasis. Galectins, expressed in tumor, stromal, and endothelial cells, contribute to thwart the development of immune responses by favoring the expansion of T regulatory cells and contributing to their immunosuppressive activity, driving the differentiation of tolerogenic dendritic cells, limiting T cell viability, and maintaining T cell anergy. The emerging data promise a future scenario in which the selective blockade of individual members of the galectin family, either alone or in combination with other therapeutic regimens, will contribute to halt tumor progression by counteracting tumor-immune escape. Here we describe a selection of methods used to investigate the role of galectin-1 in tumor-immune escape.

Regulation of galectins by hypoxia and their relevance in angiogenesis: strategies and methods.

Formation of an aberrant and heterogeneous vascular network is a key pathological event in the multistep process of tumor growth and metastasis. Pro-angiogenic factors are synthesized and released from tumor, stromal, endothelial, and myeloid cells in response to hypoxic and immunosuppressive microenvironments which are commonly found during cancer progression. Emerging data indicate key roles for galectins, particularly galectin-1, -3, -8, and -9 in the regulation of angiogenesis in different pathophysiologic settings. Each galectin interacts with a preferred set of glycosylated receptors, triggers different signaling pathway, and promotes sprouting angiogenesis through different mechanisms. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current clinical approaches. Here we describe selected strategies and methods used to study the galectin-1 regulation by hypoxia and its role in blood vessel formation.

Galectin-8: a matricellular lectin with key roles in angiogenesis.

Galectin-8 (gal-8) is a "tandem-repeat"-type galectin, containing two carbohydrate recognition domains connected by a linker peptide. gal-8 is expressed both in the cytoplasm and nucleus in vascular endothelial cells (ECs) from normal and tumor-associated blood vessels, and in lymphatic endothelial cells. Herein, we describe a novel role for gal-8 in the regulation of vascular and lymphatic angiogenesis and provide evidence of its critical implications in tumor biology. Functional assays revealed central roles for gal-8 in the control of capillary-tube formation, EC migration and in vivo angiogenesis. So far, two endothelial ligands have been described for gal-8, namely podoplanin in lymphatic vessels and CD166 (ALCAM, activated leukocyte cell adhesion molecule) in vascular ECs. Other related gal-8 functions are also summarized here, including cell adhesion and migration, which collectively demonstrate the multi-functionality of this complex lectin. Thus, gal-8 is an important component of the angiogenesis network, and an essential molecule in the extracellular matrix by providing molecular anchoring to this surrounding matrix. The implications of gal-8 in tumor angiogenesis remain to be further explored, but it is exciting to speculate that modulating gal-8-glycan interactions could be used to block lymphatic-vascular connections vital for metastasis.

Glycans and galectins in prostate cancer biology, angiogenesis and metastasis.

Prostate cancer is the second most common cause of cancer and the sixth leading cause of cancer death among men worldwide. While localized prostate cancer can be cured, advanced and metastatic prostate cancer remains a significant therapeutic challenge. Malignant transformation is associated with important modifications of the cellular glycosylation profile, and it is postulated that these changes have a considerable relevance for tumor biology. Metastasis is a multiphasic process that encompasses angiogenesis, the spread of tumor cells and their growth at distant sites from the primary tumor location. Recognition of glycoconjugates by galectins, among other lectins, plays a fundamental role in the metastatic spread, tumor immune escape and the neovascularization process. Particularly in prostate cancer, both carbohydrates and galectins have been implicated in many cellular processes such as proliferation, apoptosis, migration and invasion. However, a limited number of studies assessed their potential implications in the induction of metastasis in prostate cancer patients or in animal models. Moreover, the role of galectin-glycan interactions in vivo still remains poorly understood; concerted effort should thus be made in order to shed some light on this question. This review summarizes current evidence on both the expression and role of glycans and galectins in prostate cancer, particularly turning our attention to the angiogenic and metastatic processes.

Expression, localization and function of galectin-8, a tandem-repeat lectin, in human tumors.

Galectin-8 (Gal-8) is a 'tandem-repeat'-type galectin, which possesses two carbohydrate recognition domains connected by a linker peptide. Gal-8 complexity is related to the alternative splicing of its mRNA precursor, which is known to generate isoforms. Regarding its carbohydrate-binding specificity, Gal-8 has a unique feature among galectins, since its C-terminal domain has higher affinity for N-glycan-type branched oligosaccharides, while its N-terminal domain shows strong affinity for α2-3-sialylated or 3'-sulfated ß-galactosides. We integrate here the available information on Gal-8 expression in different tumor types and attempt to elucidate associations of its expression and localization with tumor progression with the overarching goal of analyzing its potential applications in diagnosis and prognosis. Differential diagnosis is still a prime concern in tumor pathology, and Gal-8 could be of great value in some types of primary or secondary tumors (i.e. papillary thyroid carcinoma, advanced colon carcinoma from patients with distant metastases, or metastases from primary lung carcinoma). The prognostic value of Gal-8 has been described for laryngeal carcinoma as well as advanced colon carcinoma. Further studies are needed to explain the relevance of Gal-8 and its isoforms in tumor pathology and their different intra- or extracellular roles (cytoplasmic, nuclear or extracellular) in tumor biology.

Galectins as new prognostic markers and potential therapeutic targets for advanced prostate cancers.

A better understanding of multimolecular interactions involved in tumor dissemination is required to identify new effective therapies for advanced prostate cancer (PCa). Several groups investigated protein-glycan interactions as critical factors for crosstalk between prostate tumors and their microenvironment. This review both discusses whether the "galectin-signature" might serve as a reliable biomarker for the identification of patients with high risk of metastasis and assesses the galectin-glycan lattices as potential novel targets for anticancer therapies. The ultimate goal of this review is to convey how basic findings related to galectins could be in turn translated into clinical settings for patients with advanced PCa.

Delineating the "galectin signature" of the tumor microenvironment.

Galectins, a family of glycan-binding proteins, can control tumor progression by promoting transformation, angiogenesis and immune escape. We identified a dynamically regulated 'galectin signature', which delineates the progression of prostate cancer, highlighting galectin-1 as an attractive target for anti-angiogenic therapy in advanced stages of the disease.

A unique galectin signature in human prostate cancer progression suggests galectin-1 as a key target for treatment of advanced disease.

Galectins, a family of glycan-binding proteins, influence tumor progression by modulating interactions between tumor, endothelial, stromal, and immune cells. Despite considerable progress in identifying the roles of individual galectins in tumor biology, an integrated portrait of the galectin network in different tumor microenvironments is still missing. We undertook this study to analyze the "galectin signature" of the human prostate cancer microenvironment with the overarching goal of selecting novel-molecular targets for prognostic and therapeutic purposes. In examining androgen-responsive and castration-resistant prostate cancer cells and primary tumors representing different stages of the disease, we found that galectin-1 (Gal-1) was the most abundantly expressed galectin in prostate cancer tissue and was markedly upregulated during disease progression. In contrast, all other galectins were expressed at lower levels: Gal-3, -4, -9, and -12 were downregulated during disease evolution, whereas expression of Gal-8 was unchanged. Given the prominent regulation of Gal-1 during prostate cancer progression and its predominant localization at the tumor-vascular interface, we analyzed the potential role of this endogenous lectin in prostate cancer angiogenesis. In human prostate cancer tissue arrays, Gal-1 expression correlated with the presence of blood vessels, particularly in advanced stages of the disease. Silencing Gal-1 in prostate cancer cells reduced tumor vascularization without altering expression of other angiogenesis-related genes. Collectively, our findings identify a dynamically regulated "galectin-specific signature" that accompanies disease evolution in prostate cancer, and they highlight a major role for Gal-1 as a tractable target for antiangiogenic therapy in advanced stages of the disease.