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Jun 1, 2009 (Vol. 29, No. 11)

Overcoming Protein Production Hurdles

Increasing Capabilities Becomes Essential as the Biologics Market Grows Rapidly

  • Safety and Performance

    Willem Stemmer, Ph.D., CEO, and Volker Schellenberger, Ph.D., vp drug discovery, cofounders of Amunix, discussed the concept of biosuperiors—recombinant proteins that perform better than the original pharmaceutical.

    Amunix has investigated the properties of unstructured amino acid sequences as a substitute for polyethylene glycol (PEG). While both molecules form linear, extended polymers and can be attached to proteins, PEG has a number of unsavory features, including a complex manufacturing process and a heterogeneous make-up. But, perhaps the most significant drawback to PEG is its lack of biodegradability.

    Unstructured amino acid polymers, on the other hand, do not display these adverse qualities and can be engineered for the desired length as part of a fusion molecule with the targeted biologic.

    With these concepts in mind, Amunix set out to optimize the molecule by a process that went through seven design generations over a three-year period. The optimization included in vivo half-life, expression level, genetic stability, accelerated stability, aggregation, protease resistance, and immunogenicity. Because conventional PEG is not biodegradable and accumulates to form vacuoles in the cells of the kidney, rPEG is designed to be broken down by kidney proteases, and to resist degradation for at least seven days in serum.

    Amunix has spun off a company, Versatis, to bring a number of rPEG products to market with a focus on metabolic diseases. It has worked through the manufacturing process and designed a fusion molecule, Exenatide-rPEG, for the treatment of type II diabetes that is not controlled by oral antidiabetic agents. Based on extrapolation from various animal studies, the anticipated dosing is weekly or even biweekly, Dr. Stemmer explained.

    Other projects under way use rPEG-coupled antibody fragments, including a single-chain bispecific antibody in which anti-HER-2 and anti-EGF fragments are linked together with rPEG, blocking the unwanted aggregation of the molecules while retaining the original binding ability of the molecules. Another area of investigation comprises RNAse-antibody conjugates linked together by rPEG.  

    “The rPEG gives antibody fragments long half-life and allows their expression in soluble form in the E. coli cytoplasm,” Dr. Stemmer stated. “We believe the approach holds substantial promise for the development of better therapeutic agents.”

  • Working through a Knotty Problem

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    “We approached the challenge of designing more stable protein-based drugs through the use of cystine knots,” says Jennifer Cochran, Ph.D., of Stanford University. “Also known as knottins, these cysteine-containing peptides are of small size and limited immunogenicity and make effective scaffolds for protein engineering.”

    Knottins possess disulfide-constrained loops that endow them with high thermal and proteolytic stability, and their folded protein structure is highly tolerant of multiple amino acid substitutions.

    The goal of Dr. Cochran’s group was to engineer knottin peptides that bind to alpha v integrins with high affinity and specificity for use as molecular imaging agents in living subjects.

    Integrins are a class of heterodimeric adhesion molecules that act as chemical messengers, and integrin receptors containing the alpha v subunit mediate angiogenesis and tumor metastasis. As such, they are critical players in the proliferation of tumor cells.

    The Cochran team developed a system for directed evolution of knottin peptides on the surface of yeast cells that enabled the group to identify integrin binders. Since it was known that the sequence Arg-Gly-Asp (RGD) binds to integrins, this tripeptide motif served as the basis for a combinatorial library of knottin peptides that was created and displayed on the surface of yeast. In this display format the RGD motif, constrained within a knottin loop, was surrounded by randomized bases, giving rise to around 107 different sequence possibilities. These mutants were screened in a high-throughput manner to identify knottin peptides that bound to alpha v beta 3 integrin with the highest affinities.

    The engineered integrin-binding knottin peptides bound to tumor cells with antibody-like affinities, and they blocked tumor cell attachment to extracellular matrix proteins. The knottin peptides were conjugated to a fluorescent dye or radionuclide label and were evaluated as imaging agents in glioblastoma xenograft mouse models. Peptides with higher integrin binding affinities elicited greater levels of tumor uptake compared to weaker binding peptides. Knottin peptides displayed low uptake and retention in other tissues, including the liver and kidneys, indicating their potential use as tumor targeting agents for radiotherapy or chemotherapy applications, according to the company. 

    The group’s fascination with integrins was driven by the unusual ability of these receptors to move signals in both directions: transducing information from outside the cell, and from inside-out, displaying information to the extracellular environment. This permits a rapid and flexible response on the part of the cell. Current studies are focused on the alpha v beta 3 integrin receptors and their role in regulating cell adhesion and blood vessel formation, tumor invasion, and growth and metastasis in a range of different cancers.


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