Pharma’s Cutting Edge

Pharma’s Cutting Edge
Vol. 3 Number 7 - July 2005

Protein Optimization for Therapeutic and Industrial Applications: The Next Wave in Biotherapeutics

Anyone who follows the progress in protein therapeutics is well aware of the impact made by technologies aimed at improving on nature’s protein designs. Protein conjugation with biocompatible polymers, like polyethylene glycol (PEG), called PEGylation, has extended the biological half-life, reduced immunogenicity, and altered tissue clearance of therapeutic proteins, such as interferon-alpha (Pegasys, PEG-Intron), G-CSF (Neulasta), and growth hormone analog (Somavert).

A related area of research involves changes to the proteins themselves via site-directed mutagenesis of mRNA. Proteins altered in this way are sometimes referred to as “muteins”. Altered proteins that have been successfully developed for therapeutic use thus far have been designed primarily to alter native protein pharmacokinetics. Examples of altered proteins with reduced time to maximal concentration (Tmax) include insulin aspart (NovoLog, 1 a.a. substitution in insulin) and insulin lispro (Humalog; 2 a.a. substitutions in insulin). Drugs with prolonged biological half-life and Tmax include insulin glargine (Lantus, 3 a.a substitutions in insulin) and darbepoetin alpha (Aranesp, 5 a.a. substitutions in hEPO).

One exception to the goal of altering a native protein to simply change its pharmacokinetics is pegvisomant (Somavert, 5 a.a. substitutions in hGH). This hGH analog was designed to inhibit the growth-hormone receptor. A single amino acid substitution accomplished this goal. The other alterations were made to improve the pharmacokinetics by allowing efficient PEGylation.

Probably every major pharmaceutical company and many smaller “biotech” companies are developing therapeutics and industrial proteins based on variations of the above-discussed technologies. Other successful examples of these applications—and related applications, for example, PEGylation of proteins and peptides to improve oral and pulmonary absorption—will no doubt find their way into your doctor’s office in the near future.

But what I find particularly exciting regarding the future of protein engineering is the wave of related technologies aimed at optimizing protein structure-function in a much broader variety of ways. This new wave has emerged from technical advances in high-throughput nucleic acid mutagenesis (i.e. “molecular evolution”), structural biochemistry, and novel approaches to synthetic protein synthesis and post-translational modifications. In contrast to the plethora of companies exploring traditional approaches to protein modification, relatively few companies are focused on these cutting edge protein-engineering technologies. At the risk of omitting an important member of this “protein optimization” club, I have listed here 12 companies that appear to be leading the way (Table).

You will recognize that the companies come in all sizes and flavors, from the startup (MilleGen) to the major pharmaceutical company (Lilly). You will probably also notice that the major biotechs (e.g. Genentech, Amgen) are absent. It is possible, however, that such companies are quietly pursuing related technologies.

So what is the big deal behind the protein optimization wave? The big deal is potentially big improvements over native therapies. Certainly, the successes we’ve witnessed to date are impressive, but the traditional, iterative, site-directed approaches to protein modifications are severely limited in their ability to create the variety of modifications that will be needed to take full advantage of the protein palette nature has provided.

More variety is needed to provide more consistent improvements in pharmacokinetics, targeted tissue penetration and retention, improved efficacy (e.g. increased in vivo potency), reduced adverse effects (e.g. higher receptor selectivity), and alternative delivery (anything other than parenteral would be welcome). The traditional protein engineering approaches just aren’t up to the task often enough.

The new wave can be artificially segmented into three groups: the molecular evolutionists, the structuralists, and the medicinal chemists. As you can see in the Table, the molecular evolutionists dominate the new technology wave.

The term “directed molecular evolution” was popularized by Joyce in the early 1990’s (see Joyce GF Scientific American 267(6):90-7 1992), although the concept of using processes that mimic natural evolution to create biomolecules with desired properties dates to at least the mid-1960’s. The directed molecular evolution concept is simple and can be broken down into three basic steps. Step 1: Create genetic diversity (a genetic “library”). Step 2: Screen its products for the phenotype of interest. Step 3: Make product selections. Additional steps, such as applying chemical pressure to the library to force certain phenotypes to emerge, are usually incorporated. The simplicity underlying the general approach is unfortunately mired in complex laboratory techniques whose descriptions are beyond the scope of this article. There are many review papers on the subject if you are interested.

The other camps I’ve lumped together with the molecular evolutionists, structuralists, and medicinal chemists, do not necessarily use molecular evolution techniques at all, although the three approaches are not mutually exclusive. Structuralists rely on our growing understanding of the relationships between protein sequence, higher-order structure, and function. The “protein medicinal chemists,” currently represented only by Ambrx, are attempting to create new proteins by altering the amino-acid building blocks of proteins in substantial ways.

So, which approach will prove most useful, and which company is in the best position to succeed? Too soon to say. Certainly, Lilly’s deep pockets give it a potential advantage, but such advantage will be important only if it continues to support the AME group’s R&D at its current robust levels, and that will depend on Lilly’s patience and the group’s successes. Of the private companies, Ambrx probably has the strongest financial backing, but they also have the most radical approach. I’ll keep an eye on these companies and will report on major breakthroughs when I learn of them.