 Khushboo Singh, 1st YearProgram: Chemistry Advisor: Sankaran Thayumanavan, PhD. Education: Indian Institute of Science Education and Research, Pune, BS, Chemistry (2015) |
Research Summary
A Versatile Alternate to Antibody-Drug Conjugates Cancer, second to heart diseases, is responsible for largest number of death in the US. Treatment of the disease is usually done by radiotherapy for a localized tumor or by using chemotherapy for tumors that have metastasized. Chemotherapeutic drugs kill rapidly dividing cells by targeting their cell cycle, consequently having a cytotoxic effect on normal cells lining intestinal epithelium and bone marrow. This can have severe repercussions that include nausea, pain, cardiotoxicity, hair loss and impaired immune system. A promising strategy in cancer therapy is aimed at achieving active targeting using antibody-drug conjugates (ADCs). ADCs are comprised of monoclonal antibodies conjugated to a cytotoxic agent via a linker. Monoclonal antibodies are used to target an antigen that is over-expressed on the surface of tumor cells. ADCs bind to the tumor specific antigen and are internalized by the cell through receptor mediated endocytosis. A major challenge in development of antibody-drug conjugates is in the design of an appropriate linker that remains responsive to intra-cellular condition of a cell such as reducing environment, acidic environment or presence of certain enzymes (cathepsin) but maintains extra-cellular stability of ADC. ADCs must function as a pro-drug releasing the active drug only when the linker is cleaved inside target cells. Secondly, the number of molecules of drugs conjugated to an antibody ranges from 1-8 with an average of 3.5. Increasing the number of drug molecules on the antibody can impair its systemic circulation half-life and reduce antigen binding capability. The two major limitations of ADCs, developing an ideal linker for drug conjugation and low drug to antibody ratio, can be overcome by using polymeric nanoparticles. The cytotoxic drug can be physically encapsulated in the polymeric carrier excluding the need for covalent modification on the drug itself. The active targeting of ADCs can be retained by conjugating the antibody on the polymer particle thereby developing antibody-nanoparticle conjugates. Since antibodies only serve as a targeting agent, it reduces the amount of antibody needed for conjugation and greatly minimizes the cost of therapy. Immunotherapy for cancer is an emerging field in biotechnology and is proving to be better than small molecule drugs alone. My research derives motivation from this technology and is aimed at developing a platform for conjugation of a monoclonal antibody (mAb), specific for a tumor antigen, on to a polymeric nanogel composed of polyethylene glycol (PEG) as the hydrophilic unit and pyridyl disulfide (PDS) as the hydrophobic unit. The selectivity of these antibody conjugated nanogels towards CD4+ cells has already been shown by a senior in Thayumanavan/ Osborne Lab. Nonetheless, conjugating the whole antibody on the nanoparticle proved to be a daunting task. An antibody is composed of two light chains and two heavy chains, that are connected at the hinge by disulfide linkages to yield a Y-shaped protein that weighs about 150 kDa. Since it is only the Fab’ region of the antibody that imparts antigenm recognition, I have developed a strategy to obtain Fab’ domain of mAb, essentially eliminating the need for conjugation of a whole antibody on the nanogel. Working in collaboration with Osborne lab, I have shown that these Fab’ domains retain their specificity towards CD4+ cells. My plan is to conjugate these Fab’ (MW ~55 kDa) to the nanogel using thiol-specific bio-orthogonal reactions (sulfhydryl with monobromomaleimide) following which the antibodynanogel conjugate will be tested for its in-vitro and in-vivo targeting capability. Furthermore, the bio-distribution and the pharmacokinetics of these nanoparticles will be studied in-vivo. The biotech world has seen tremendous efforts in the development of many antibody based therapies and the relevance of this research to my work has inspired me in exploring this field further.