analyzing-tumor-tissue-distribution-target-antigen-specific

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

University of Virginia School of Medicine

In mammals, gene expression is largely controlled at the transcriptional level. In response to environmental or intrinsic signaling, gene expression is often fine-tuned by epigenetic modifications, including DNA methylation and histone modifications. One such histone modification is ubiquitination that predominately occurs in mono-ubiquitinated forms on histone H2A and H2B. We recently identified and characterized a novel E3 ligase called TRIM37 that ubiquitinates H2A. This study highlights the consequence of aberrant histone ubiquitination at the promoters of tumor suppressor genes in breast cancer. Regulatory mechanism by which TRIM37 and other auxiliary proteins are involved in the initiation and progression of breast cancer is of utmost importance toward generating effective therapeutics. Here, we describe a detailed step-by-step process of carrying out in vitro ubiquitination assay using purified histone proteins or reconstituted nucleosomes and affinity-purified recombinant E3 ligase like TRIM37. These experimental procedures are largely based on our studies in mammalian cells and will be a useful tool to identify substrate for E3 ubiquitin ligase as well as characterizing new E3 ligases.

In Vitro Assay to Study Histone Ubiquitination During Transcriptional Regulation.

Generating effective RNAi-based therapies with the potential to achieve leukemia remission remains critical unmet need. Despite a growing number of leukemia clinical trials, tissue specific delivery of therapeutic siRNA is a major roadblock in translating its clinical potential. The most recent reports in the antibody-siRNA-conjugates (ARCs) field add new dimensions to leukemic therapy, where a covalently ligated therapeutic antisense-RNA with the potential to repress the oncogenic transcript is selectively delivered into the cancer cells. Despite ARC localization to leukemic cells due to high affinity antigen-antibody interactions, multiple challenges exist to unlock the therapeutic potential of siRNA targeting. Areas covered: This review focuses on antibody and siRNA-based therapies for leukemia as well as potential antibody engineering-based strategies to generate an optimal ARC platform. Expert opinion: In vitro and clinical results have revealed that non-targeted delivery and inefficient cellular internalization of therapeutic siRNA are major contributing factors for the lack of efficacy in leukemia patients. Rational antibody design and selective protein engineering with the potential to neutralize siRNA charge, stabilize ARC complex, restrict off-targeted delivery, optimize endosomal escape, and extend serum half-life will generate clinically relevant leukemic therapies that are safe, selective, and effective.

Antibody-siRNA conjugates: drugging the undruggable for anti-leukemic therapy.

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked  in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked  in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked  in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked  corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked  in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked  in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked  as a therapeutic approach for RTT.

Pharmacological reactivation of inactive X-linked  in cerebral cortical neurons of living mice.

Therapeutic antibodies targeting ovarian cancer (OvCa)-enriched receptors have largely been disappointing due to limited tumor-specific antibody-dependent cellular cytotoxicity. Here we report a symbiotic approach that is highly selective and superior compared with investigational clinical antibodies. This bispecific-anchored cytotoxicity activator antibody is rationally designed to instigate "cis" and "trans" cytotoxicity by combining specificities against folate receptor alpha-1 (FOLR1) and death receptor 5 (DR5). Whereas the in vivo agonist DR5 signaling requires FcγRIIB interaction, the FOLR1 anchor functions as a primary clustering point to retain and maintain a high level of tumor-specific apoptosis. The presented proof of concept study strategically makes use of a tumor cell-enriched anchor receptor for agonist death receptor targeting to potentially generate a clinically viable strategy for OvCa.

Article	Year
In Vitro Assay to Study Histone Ubiquitination During Transcriptional Regulation. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27832544	2017
Antibody-siRNA conjugates: drugging the undruggable for anti-leukemic therapy. Expert opinion on biological therapy\| PubMed ID: 27977315	2017
Pharmacological reactivation of inactive X-linked in cerebral cortical neurons of living mice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30012595	2018
A Single-Agent Dual-Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer. Cancer cell\| PubMed ID: 30107179	2018

Jogender Tushir-Singh

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