Targeting miRNA by CRISPR/Cas in cancer: advantages and challenges.

Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade. In the study of cancer, the CRISPR/CRISPR-associated protein (Cas) system opens new avenues into issues that were once unknown in our knowledge of the noncoding genome, tumor heterogeneity, and precision medicines. CRISPR/Cas-based gene-editing technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs (miRNAs). However, the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities. This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy. Furthermore, we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.

The interaction between miRNAs/lncRNAs and Notch pathway in human disorders.

Notch pathway is a signaling cascade with important impacts on cell proliferation, differentiation, developmental processes and tissue homeostasis. This pathway also regulates stem cell properties, thus being involved in both normal developmental processes and metastatic capacity of cancer cells. Lots of lncRNAs and miRNAs have been recognized that control Notch pathway at some levels or their expression is regulated by this pathway. FOXD2-AS1, MEG3, ANRIL, linc-OIP5, lincRNA-p21, CBR3-AS1, HOTAIR, PVT1 and GAS5 are among lncRNAs that interact with Notch signaling. miR-19, miR-21, miR-33a, miR-8/200, miR-34a, miR-146a, miR-37, miR-100, miR-107 and several other miRNAs have functional interplay with this signaling cascade. In the present review article, we have illuminated the interplay between lncRNAs/miRNAs and Notch pathway in two distinct contexts i.e. cancers and non-neoplastic conditions.

Unconventional immunotherapy with an unconventional target.

Pritumumab, a natural human IgG1 kappa antibody was obtained from a regional draining lymph node of a patient with cervical carcinoma through traditional hybridoma technology. Specificity analysis of the target antigen, an altered form of vimentin called, ecto-domain vimentin (EDV), shows it to be limited to cell surface expression on cancer cells. Clinically, 249 brain cancer patients were treated with a low dose pritumumab regimen, either at 1 mg once a week or 1 mg twice a week, and of those evaluated overall response rates of between 25-30% were seen with several complete and partial responses. A clinical trial assessing higher doses of pritumumab as a therapeutic for brain cancer is expected to begin this year. Overall, these data together suggest pritumumab is suitable for further development as an anti-tumor therapeutic.

Brain-derived neurotrophic factor downregulation in gastric cancer.

The brain-derived neurotrophic factor (BDNF) is a certain type of growth factor that participates in the correct construction of the brain. Moreover, some reports have shown its participation in the tumorigenesis process. A long noncoding RNA known as BDNF-antisense (BDNF-AS) is shown to be transcribed from the antisense direction of the BDNF gene and control its expression. In the current study, we compared expression levels of BDNF and its antisense in gastric cancer tissues and adjacent noncancerous tissues (ANCTs) using quantitative real-time polymerase chain reaction. Expression of both genes tended to decrease in gastric cancer tissues in comparison with ANCTs (expression ratio = 0.4 and P = .06 for BDNF; expression ratio = 0.35 and P = .05 for BDNF-AS, respectively). Relative transcript levels of both genes were remarkably associated with the site of primary tumor in a way that all cardia tumors had low levels of both BDNF and BDNF-AS in comparison with their paired ANCTs (P = .002 and P = <.001). We also found higher amounts of both genes in malignant samples obtained from older patients (P = .01 and P = .03 for BDNF and BDNF-AS, respectively). Besides, BDNF expression was higher in tumors with lymphatic/vascular invasion (P = .01). There was also a trend toward upregulation of BDNF-AS in tumors with lymphatic/vascular invasion (P = .05). The current study underscores the role of BDNF and BDNF-AS in the pathogenic process leading to gastric cancer.

Hepatocellular carcinoma up-regulated long non-coding RNA: a putative marker in multiple sclerosis.

Highly up-regulated in liver cancer (HULC) is a cancer-associated long non-coding RNA (lncRNA) which may regulate expression of other genes by working as a competing RNA for microRNAs. In the current study, we assessed transcript levels of this lncRNA in peripheral blood of multiple sclerosis (MS) patients and healthy persons to evaluate its possible role in the pathogenesis of this inflammatory disease and its diagnostic power. The results of Multilevel Bayesian showed no significant difference between cases and controls (P = 0.002, 95% confidence interval (CI) = [3.08, 13.3]). However, based on the results of Quantile regression, there was a significant difference in HULC expression between cases and controls after controlling the effects of sex and age (P = 0.002, 95% CI = [3.08, 13.3]) which shows different trends in males and females. HULC expression was inversely correlated with age of male subjects but not female subjects. HULC transcript levels had 91.1% accuracy in diagnosis of MS disease (Specificity: 80%, Sensitivity: 86.6%). The diagnostic power of HULC was higher in male subjects aged less than 50 years (AUC = 0.923, Specificity: 80%, Sensitivity: 100%). The present study shows the possibility of application of transcript levels of HULC as diagnostic marker in MS disease. However, future studies with larger sample sizes are necessary to validate our results.

Antibody drug conjugates: Progress, pitfalls, and promises.

Antibody drug conjugates (ADCs) represent a promising and an efficient strategy for targeted cancer therapy. Comprised of a monoclonal antibody, a cytotoxic drug, and a linker, ADCs offer tumor selectively, reduced toxicity, and improved stability in systemic circulation. Recent approvals of two ADCs have led to a resurgence in ADC research, with more than 60 ADCs under various stages of clinical development. The therapeutic success of future ADCs is dependent on adherence to key requirements of their design and careful selection of the target antigen on cancer cells. Here we review the main components in the design of antibody drug conjugates, improvements made, and lessons learned over two decades of research, as well as the future of third generation ADCs.

A Binding Potency Assay for Pritumumab and Ecto-Domain Vimentin.

Pritumumab, a natural human IgG1kappa mAb, was isolated from the regional lymph node of a patient with cervical cancer. This antibody has been reported to bind the cytoskeletal protein vimentin, and to cell surface expressed vimentin referred to as ecto-domain vimentin (EDV). Here, we report details of the development of a potency of binding assay for pritumumab as a prerequisite before pursuing clinical trials. The enzyme linked immunosorbent assay (ELISA) to detect antibody-binding antigen can serve as a potency assay for release of manufactured samples to be used in clinical studies. Several layers of controls for this assay along with suitability testing for reagents and components of the assay must be developed before the assay can be incorporated for stability testing and release of manufatured samples.

Pritumumab, the first therapeutic antibody for glioma patients.

Immunotherapy is now at the forefront of cancer therapeutic development. Gliomas are a particularly aggressive form of brain cancer for which immunotherapy may hold promise. Pritumumab (also known in the literature as CLNH11, CLN-IgG, and ACA-11) was the first monoclonal antibody tested in cancer patients. Pritumumab is a natural human monoclonal antibody developed from a B lymphocyte isolated from a regional draining lymph node of a patient with cervical carcinoma. The antibody binds ecto-domain vimentin on the surface of cancer cells. Pritumumab was originally tested in clinical trials with brain cancer patients in Japan where it demonstrated therapeutic benefit. It was reported to be a safe and effective therapy for brain cancer patients at doses 5-10 fold less than currently approved antibodies. Phase I dose escalation clinical trials are now being planned with pritumumab for the near future. Here we review data on the development and characterization of pritumumab, and review clinical trails data assessing immunotherapeutic effects of pritumumab for glioma patients.

Summary analysis of the pre-clinical and clinical results of brain tumor patients treated with pritumumab.

Pritumumab is a human IgG1 kappa antibody that has been derived from a B-cell isolated from a regional draining lymph node of a patient with cervical carcinoma. Specificity analysis of the antibody with human tissues showed the antigen, altered tumor-associated vimentin, to be highly restricted to various cancers and not normal cells and tissues. In various clinical trials in Japan 249 patients with brain cancer were treated with pritumumab. The overall response rate was between 25-30% with several survivors beyond 5-years post-treatment. The patients were on a low dose regimen of 1mg given twice a week for a course of 24 weeks for a total dose of 48 mgs per course. Pritumumab appears to be a safe and effective therapy in patients with malignant gliomas.

Cocktails of human anti-cancer antibodies show a synergistic effect in nude mouse tumor xenografts.

A panel of four natural human monoclonal IgG antibodies derived from B lymphocytes isolated from regional draining lymph nodes of cancer patients has been developed and characterized. The four human antibodies are termed, RM1, RM2, RM3, and RM4. The immunoreactivity of this panel of four human antibodies is restricted to tumor cells. Individually, these human MAbs show tumor targeting and are effective in inhibiting tumor growth in nude mouse xenograft models. When used in combination the antibodies show an additive effect in slowing down the progression of tumors in xenograft models suggesting that cocktails of antibodies may be useful in the clinic.

Requirements for human antibody cocktails for oncology.

Cancer patients receiving antibodies as monotherapy have benefited from these treatments. However, significant improvements can be made that should make the therapy more effective. Applying lessons learned from the natural oligoclonal antibody response that cancer patients mount to their own tumours suggests that cocktails of monoclonal antibodies could be formulated, which may be more effective in treating cancers. The next phase of antibody immunotherapy will include cocktails of monoclonal antibodies. Various requirements for human antibody cocktails are discussed, as well as potential limitations of this approach.

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.