Monoclonal and Polyclonal Antibodies Specific to Human Fibromodulin.

BACKGROUND: The unique expression of fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL) has been previously reported. Detecting FMOD in CLL patients using specific anti-FMOD mAbs might provide a promising method in detection, monitoring, and prognosis of CLL. OBJECTIVES: In this study, we aimed for producing specific antibodies against FMOD to facilitate further cohort study of CLL, thus addressing FMOD as a potential target of detection. MATERIALS AND METHODS: Human FMOD gene (1087 bp) was extracted from genome of the CLL patients, and was cloned into the expression vector of pET-22b (+). The recombinant FMOD protein (rFMOD) was expressed in Escherichia coli. The purified rFMOD protein was used as an immunogen in rabbit and mice. Hybridoma technology was used to develop the monoclonal antibodies (mAbs). Polyclonal antibody (pAb) was purified from the rabbit sera using affinity column. The reactivity of anti-FMOD antibodies was assessed in ELISA, immunocytochemistry (ICC) and Western blot. RESULTS: ICC results showed that the anti-FMOD antibodies specifically detected FMOD in CLL PBMCs and cell lines. The developed anti-FMOD pAb detected FMOD in CLL lysates, compared to healthy PBMCs, in Western blot and ELISA. CONCLUSIONS: The developed anti-FMOD mAbs, and pAb specifically detect FMOD in CLL samples and might be used as research tools for further investigations in CLL.

Anchored Fibromodulin as a Novel Target in Chronic Lymphocytic Leukemia: Diagnostic and Therapeutic Implications.

BACKGROUND: We have previously reported the aberrant expression of Fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). Although FMOD has been considered as a cytoplasmic or secretory protein, we discovered the cell surface expression of FMOD in leukemic B cells via anchoring with glycosylphosphatidylinositol (GPI). OBJECTIVE: To evaluate FMOD as a new biomarker in CLL patients in comparison with healthy individuals. METHODS: A monoclonal antibody was generated against human FMOD. The cell surface expression of FMOD in 52 CLL patients and 45 healthy individuals were compared by flow cytometry. A bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) was used to determine the cell surface localization of FMOD using ELISA and flow cytometry techniques. Annexin V-FITC and propidium iodide (PI) was used to detect apoptosis induction in CLL PBMCs following in vitro incubation with anti-FMOD mAb. RESULTS: The results demonstrated the widespread cell surface expression of GPI-anchored FMOD in CLL patients (median: 79.9 %), although healthy individuals had low FMOD expression (median: 6.2 %) (p≤0.0001). The cut-off value of FMOD expression was estimated with high sensitivity and specificity at 17.9 %. Furthermore, in vitro apoptosis induction of leukemic cells following incubation with anti-FMOD mAb showed a direct apoptosis of CLL cells (27.9%) with very low effect on healthy PBMCs (6%). CONCLUSION: The membrane-anchoring of FMOD by means of a GPI moiety in leukemic cells supports FMOD as a highly potential diagnostic and therapeutic target in CLL patients.

Sortilin as a Novel Diagnostic and Therapeutic Biomarker in Chronic Lymphocytic Leukemia.

BACKGROUND: The overexpression of sortilin/neurotensin receptor 3 has previously been reported in various human solid tumors but not in hematological malignancies. Here, we report the overexpression of sortilin in leukemic cells from patients with Chronic Lymphocytic Leukemia (CLL). METHODS: Flow cytometry was used to compare the expression of sortilin in CLL patients (n=52) and healthy individuals (n=26). Also, in vitro apoptosis induction was assessed in CLL Peripheral Blood Mononuclear Cell (PBMCs) following directly targeting of sortilin. RESULTS: The results showed a significant expression of sortilin on the surface of CLL PBMCs (range from 2.2 to 71.5%) in comparison to healthy individuals (range from 0.03 to 7.4%) (p≤0.0001). The optimal cut-off value of sortilin expression was determined at 7.2% with high sensitivity and specificity. Treatment of leukemic cells with anti-sortilin antibody could induce apoptosis without any effect on normal cells. CONCLUSION: Apoptosis induction in CLL cells together with a significant correlation between the expression of sortilin and CD23 represent a possible functional role of sortilin in leukemogenesis of CLL cells. Therefore, sortilin might be considered as a promising novel biomarker in diagnosis, monitoring, and therapy of patients with CLL.