Effect of Chlorella vulgaris on Liver Function Biomarkers: a Systematic Review and Meta-Analysis.

This study presents a comprehensive systematic review and meta-analysis of randomized controlled trials (RCTs) on Chlorella vulgaris (C. vulgaris) supplementation and liver function biomarkers. Pertinent studies were identified using Scopus, ISI Web of Science, PubMed, and Cochrane library databases up to August 2020. Mean differences were pooled using a random-effects model. Pooling 7 RCTs together showed that C. vulgaris supplementation led to a significant reduction of serum aspartate aminotransferase (AST) levels (weighted mean difference [WMD], -9.15 U/L; 95% confidence interval [CI], -16.09, -2.21), but not alanine aminotransferase (ALT) or alkaline phosphatase (ALP) levels compared to the placebo consumption. Subgroup-analysis indicated that C. vulgaris supplementation had more effect on AST decreasing among non-alcoholic fatty liver disease patients (WMD, -16.42 U/L; 95% CI, -29.75, -3.09) than others. Furthermore, subgroup analysis based on kind of compression showed that C. vulgaris supplementation significantly decreased ALT levels (WMD, -4.65 U/L; 95% CI, -8.88, -0.42) compared with the placebo, but not metformin consumption. It seems that C. vulgaris supplementation mainly affects AST levels rather than ALT and ALP levels, however, as mentioned the effect of C. vulgaris on those enzymes might be context-dependent. Therefore, further investigations with a large number of patients as well as on different disorders are necessary and can provide more definitive evidence.

Development of a Novel Inhibitory Chimeric Anti-HER2 Monoclonal Antibody.

BACKGROUND: We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. OBJECTIVE: To describe chimerization and functional characterization of 2A8 mAb. METHODS: The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. RESULTS: Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. CONCLUSION: The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.

Differential Effects of Inhibitory and Stimulatory Anti-HER2 Monoclonal Antibodies on AKT/ERK Signaling Pathways

Objective: Homo- and heterodimerization of the receptor tyrosine kinase HER2 hyperactivate several downstream signaling pathways, leading to uncontrolled growth and proliferation of tumor cells. Anti-HER2 monoclonal antibodies (mAbs) may induce different effects on HER2 dimerization and signaling. Methods: The effect of two inhibitory (2A8, 1T0) and one stimulatory (1H9) anti-HER2 mAbs either alone or in combination with trastuzumab was investigated on AKT and ERK signaling pathways and HER2 degradation in a human breast cancer cell line (BT-474) by Western blotting. Result: While 1H9 mAb had no significant effect on AKT and ERK signaling pathways, 1T0 and 2A8 mAbs inhibited phosphorylation of both pathways. Combination of 1T0 mAb with trastuzumab resulted in significant synergistic inhibition of both pathways and HER2 degradation, much more potently than the combination of trastuzumab and pertuzumab. Conclusion: Our data indicate that anti-HER2 mAbs may induce different signaling pathways depending on their effect on tumor cell growth and proliferation. The significant inhibition of ERK and AKT phosphorylation by 1T0 alone or particularly in combination with trastuzumab suggests its potential therapeutic application for targeted immunotherapy of HER2 overexpressing malignancies.

Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition.

Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.

Inhibition of tumor growth by mouse ROR1 specific antibody in a syngeneic mouse tumor model.

INTRODUCTION: Immunotherapy with tumor-associated antigens (TAAs) is a potentially powerful approach to eradicate tumor cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) plays a crucial role for survival of tumor cells and is overexpressed in various malignancies. In the present study, we developed a syngeneic mouse tumor model to assess anti-tumor effect of mouse ROR1 specific polyclonal antibody (pAb) in vivo. MATERIALS AND METHODS: Mouse ROR1 specific antibody was produced in rabbit using recombinant ROR1 protein. Tow mouse tumor cell lines, (4T1 and CT26), were transfected with full length mouse ROR1 construct and stable clones were selected and characterized by immunocytochemistry, Western blot and flow cytometry. In vitro and in vivo anti-tumor activities of anti-ROR1 antibody were assessed by XTT and syngeneic BALB/c mouse model, respectively. RESULTS: We successfully established two mouse ROR1-overexpressing tumor cell lines. The in vitro results indicate that the ROR1pAb did not significantly inhibit growth of ROR1+ cell lines. One of these cell lines (CT26-ROR1) was implanted in syngeneic BALB/c mice to assess anti-ROR1 tumor inhibitory activity in vivo. The tumor size was significantly reduced in mice treated with ROR1 specific pAb. CONCLUSION: Our results demonstrated for the first time tumor inhibitory effect of mouse ROR1 specific antibody in a syngeneic mouse tumor model. This model is a promising tool for preclinical assessment of ROR1 therapeutics and investigation of the underling molecular mechanisms.

Epitope Mapping of Human HER2 Specific Mouse Monoclonal Antibodies Using Recombinant Extracellular Subdomains

Background: Human epidermal growth factor receptor 2 (HER2) is overexpressed in several human malignancies and numerous studies have indicated that it plays important roles in the development and maintenance of the malignant phenotype. Targeting of HER2 molecules with monoclonal antibodies (mAbs) is a promising therapeutic approach. However, anti-HER2 mAbs affect cancer cells differently, depending on the distinct epitopes which are the targets. Methods: Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues. Results: Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one reacted with subdomain I+II, 2 recognized either subdomain III or IV and 2 recognized subdomain III+IV. However, none of our mAbs recognized the subdomain II alone. The mAbs displayed either inhibitory or stimulatory effects on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implied better reactivity of the mAbs recognizing linear epitopes. Conclusions: Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, there seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.

Polyclonal Antibody against Different Extracellular Subdomains of HER2 Induces Tumor Growth Inhibition in vitro.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has a crucial role in several malignancies. The extracellular domain of HER2 (HER2-ECD) has been extensively employed as an important target in passive and active immunotherapy. Isolated recombinant prokaryotic HER2-ECD subdomains were previously found to be ineffective in inducing anti-tumor antibody response. OBJECTIVE: To employ recombinant eukaryotic HER2-ECD subdomains to raise anti-HER2 antibodies and determine their anti-tumor activity in vitro. METHODS: Two paired subdomains of HER2-ECD (DI+II and DIII+IV), representing Pertuzumab and Trastuzumab binding domains, respectively, along with the full extracellular domain of HER2 were generated in CHO-K1 cells. Polyclonal antibodies were raised against these subdomains and characterized using ELISA, flow cytometry, and immunoblot and their anti-tumor activity was assessed by XTT assay. The cross-reactivity of these antibodies was specified along with other members of the human HER family. RESULTS: Similar to Trastuzumab and anti-HER2-ECD antibody, anti-DI+II and DIII+IV polyclonal antibodies reacted with recombinant HER2-ECD and native HER2 expressed on tumor cells. These two polyclonal antibodies were able to inhibit the binding of Pertuzumab and Trastuzumab to HER2, respectively, and did not cross-react with other members of HER family. These antibodies were able to inhibit tumor cell growth in vitro, similar to Trastuzumab. CONCLUSION: The high immunogenicity of human HER2 DI+II and DIII+IV subdomains in rabbits and the tumor inhibitory activity of the purified specific antibodies imply that they might be suitable for active immunotherapy in formulation with appropriate adjuvants and in combination with other HER2 specific therapeutics.