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Sep 15, 2013 (Vol. 33, No. 16)

Progress in Protein Expression Technology

  • Combining Synthetic Antibody Technologies and Structure-Based Design

    Click Image To Enlarge +
    Researchers at the University of Toronto constructed a synthetic antigen-binding fragment library designed to directly compare the contributions that CDR-H3 and CDR-L3 make to antigen recognition. They used a single optimized human framework and diversified the three heavy-chain CDRs and CDR-L3.1. The backbones of the heavy- and light-chain variable domains are shown as tubes. The frameworks are colored gray and the CDR loops are colored as follows: CDR-L3 (purple), CDR-H1 (yellow), CDR-H2 (orange) and CDR-H3 (red). [Jarrett Adams/University of Toronto]

    “Our main goal is to generate proteins that are well folded and can be used for in vitro selection, but most large proteins do not always fold well and may be unstable,” said Sachdev Sidhu, Ph.D., professor at the Banting and Best Department of Medical Research, University of Toronto. To overcome this limitation, a strategy that Dr. Sidhu and colleagues are using is to perform structural analysis to identify smaller, structured protein domains, which can be expressed as a native fold that can be used as a scaffold to engineer desired binding specificities against full-length, endogenous proteins. The ability of these smaller, engineered domains to bind full-length, native cellular proteins is subsequently used to assess their functionality.

    A key research effort in Dr. Sidhu’s lab is the synthesis of recombinant antibodies in bacterial and mammalian expression systems. “Bacteria have the advantage of growing very rapidly and enabling the construction of large combinatorial libraries, and after using phage display to perform engineering and design, we use mammalian systems to produce full-length antibodies, which are the closest to the natural ones,” said Dr. Sidhu.

    Tailored mutagenesis facilitates the engineering of functional antigen-binding sites with reduced hydrophobicity, which in turn helps generate stabilized, less aggregation-prone proteins. “This illustrates the advantage of combining synthetic antibody technologies with structure-based design,” said Dr. Sidhu.

    By using this strategy, Dr. Sidhu and collaborators identified antibodies that target uncleaved and cleaved Ebola virus envelope glycoprotein, and dynamically characterized viral fusion and entry, key steps during viral pathogenesis. “The bigger challenge in biology is to generate antibodies to antigens that are more difficult to express, such as membrane proteins, and developing those expression systems is currently the limiting factor for us and for a lot of groups,” said Dr. Sidhu.

  • Single-Domain Antibodies

    “As a research lab, we have been involved in basic discovery and translational science, and making the transition to commercial use is currently one of the major gaps in the field,” said Mitchell Ho, Ph.D., chief of the antibody therapy section at the National Cancer Institute. Dr. Ho and colleagues are focused on the development of therapeutic antibodies for several types of malignant tumors. This process relies on phage display in E. coli, followed by the transition to mammalian expression systems to generate the constructs of interest.

    “In recent years, we became particularly interested in single-domain antibodies,” said Dr. Ho. The lack of the immunoglobulin light chain provides single-domain antibodies with unique functional characteristics because the heavy chains are able to better recognize unique and novel hidden epitopes that would not be accessible for whole immunoglobulin G molecules.

    Research efforts in Dr. Ho’s laboratory led to the synthesis of a human heavy-chain, variable-domain antibody, HN3, against glypican-3, which promises to provide a new therapeutic approach for liver cancer. Additionally, Dr. Ho and colleagues recently reported the synthesis of SD1, the first human single-domain antibody that targets tumors expressing mesothelin, a plasma membrane differentiation antigen. This could improve the therapeutic prospects of patients with mesothelin-expressing tumors, which include mesothelioma, ovarian cancer, and pancreatic and lung adenocarcinoma.

    Current efforts in Dr. Ho’s laboratory are focused on synthesizing immunocytokines, which consist of antibodies fused to cytokines and are promising therapeutic candidates. “For some of these immunocytokines, expression in mammalian systems is too low, but baculovirus expression systems in insect cells work well, and we showed that the products, in addition to being expressed at high levels, are soluble and active,” said Dr. Ho.

  • Enhancing Expression Systems

    “Significant improvements that we have seen in biologics manufacturing over the years are based on advances in cell-line development and media/process development, but also due to the focus of such efforts on a single cell substrate platform, CHO cells,” said Shyamsundar Subramanian, Ph.D., principal scientist and head of vaccine expression systems and process development, Merck & Co. “Unfortunately, no universal platforms are available yet for vaccines, even if one is restricted to protein antigens.”

    Partly due to the variety and diversity of vaccine antigens, this shortcoming creates challenges in the a priori choice of cell substrates, and thus requires the systematic and efficient screening of expression platforms for vaccine antigens. Dr. Subramanian and colleagues have, in the past few years, focused on identifying the most suitable expression systems for vaccine candidates.

    The systematic analysis of several expression systems for expressing virus-like envelope particles and bacterial toxins from Clostridium difficile revealed that insect cell platforms are the most successful. “This is the first time that Merck is using insect cells to produce human vaccine candidates,” said Dr. Subramanian. Vaccine candidates such as virus-like particles and bacterial toxins exhibit some levels of cytotoxicity in mammalian expression systems. Moreover, the use of bacterial expression systems can impact product quality and purifiability.

    Baculovirus-mediated, transient overexpression in insect cells has turned out to be a particularly attractive option. One of the key lessons from the expression of various antigens is that certain expression systems work better, depending on the protein of interest. “We cannot expect a single expression system to always succeed, but we are trying to focus our attention on a few platforms that are promising,” said Dr. Subramanian.

  • Hastening Biomedical Progress

    Protein expression has become one of the most frequently performed experimental procedures in the life sciences. The synthesis of proteins or protein fragments is a virtually indispensable ingredient of most biomedical endeavors, and represents a critical determinant of their much-anticipated success. With the increasing number of available genomes, and the vast datasets that can be generated with the omics platforms, we can expect that interest in more robust and reliable protein expression, purification, and characterization approaches will only grow in the years to come.

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