No biotech-based drug has busted more blocks than recombinanterythropoietin, better known as EPO. This Amgen Inc.-createdrecombinant blood-enhancer generated some $2.6 billion in salesworldwide in 1995.

That number is two to three times higher than revenues grossed bythree runner-up blockbuster pharmaceuticals, developed byGenentech Inc. _ human growth hormone (Protropin and Nutropin)and tissue plasminogen activator (Activase).

Amgen's spokesman, David Kaye, told BioWorld Today that 1995sales of EPO in the U.S. totaled $885 million last year.

FDA approved Amgen's EPO on June 1, 1989, to treat the chronicanemia suffered by patients on kidney dialysis. It now is alsoprescribed for the side-effects of AZT in treating HIV infection.

Amgen has licensed certain markets for its EPO patents to OrthoBiotech Inc., of Raritan, N.J., a subsidiary of Johnson & Johnson, ofNew Brunswick, N.J.

One crypto-outlet for EPO is thought to be athletes, who reportedlyshoot up the blood-boosting hormone to get rushes of energy andendurance. Unlike other such illicit aids to physical performance,recombinant EPO escapes detection in testing, because its moleculecan't be told apart from the natural erythropoietin the body secreteswhen it feels the need for more oxygen in its tissues.

EPO stimulates the production of red blood cells (RBC), which carryoxygen-rich hemoglobin. It does this by activating RBC's erythroidprogenitor cells in bone marrow and blood. EPO itself is made in thekidneys.

In charge of this metabolic process are the erythropoietin receptors,which the hormone turns on by binding to contact points on theirbroad surface.

Cutting A Compound Down To Size

EPO is a big, bulky protein, so Johnson & Johnson scientists set outto find a smaller peptide, which could mimic this receptor-triggeringeffect. At the R.W. Johnson Research Institute, in Raritan, molecularbiologist Linda Joliffe is co-team leader of this project.

She also is a co-author of a research article in today's Science, titled:"Small peptides as potent mimetics of the protein hormoneerythropoietin."

What she and her colleagues knew going into this project, Joliffe toldBioWorld Today, was that "even though the large molecule contactsa large surface area on the receptor, not all of those contacts areequal. In other words, some of those points are responsible for thebinding energy that holds the [EPO] ligand on the receptor."

The Johnson institute began this project in 1989. "To try to get asmaller molecule that could activate the receptor," Joliffe recalled,"we approached the Affymax Research Institute in Palo Alto, Calif.They used a phage-display technique to discover the peptide."

Molecular and cell biologist Nicholas Wrighton, of Affymax, ofAmsterdam, the Netherlands, is the Science paper's first author. Toseek out a compound unrelated to EPO but which could bind to itsreceptor, he told BioWorld Today, "We screened tens of phage-basedlibraries, many of which had 109 [a billion billion] compounds, andfound one hit."

When they synthesized it, Wrighton continued, "It was a fairly low-affinity-binding peptide." Then they assayed its ability to prod EPOreceptors into making the human hormone grow in a cell linetransfected with the receptor. The cells respond to EPO by growing,but "essentially, these peptides seemed inactive; they didn't make thecells grow."

Whereupon, the Affymax team mutagenized their reluctant peptideand developed a large family of mutant compounds, about 100sequences. When they tested this second-generation peptide in thecell proliferation assay, "the cells grew. And the parental cell line,which did not have the receptor, did not grow."

Wrighton emphasized that not only did the peptide mimic the effectsof erythropoietin but it did so without the slightest sequencesimilarity to the EPO molecule. "In my hands, in the cell proliferationassay," he said, "the neutralizing antibody inhibited EPO, but had noeffect on the peptide. Therefore, we hadn't inadvertently put EPO inthere."

This scruple, he explained, prevailed "because EPO is patented byAmgen. I guess they have a say on that sequence."

Recombinant EPO is a cytokine hormone 165 amino acids long, witha molecular weight of 34,000. The peptide mimic has only 20 aminoacids, and a molecular weight of 2,100.

A `Very Surprising Result'

"For some reason, and we don't know why," Wrighton went on, "thepeptide dimerizes," joining two of its molecules to bind to tworeceptors. "It's a very surprising result," he observed. "We believeit's the mechanism by which the peptide attaches to selected contactpoints on the receptor, which it then activates."

Back in Raritan, Johnson investigators "showed that the peptidecould generate red blood cell precursor colonies, just as EPO does,"Wrighton said.

Then, structural biologists at the Scripps Research Institute in LaJolla, Calif. co-crystallized the receptor with a synthetic peptide, toreveal in atomic detail how the peptide binds and dimerizes to theEPO receptor.

Their report appears in today's Science in a separate paper titled:"Functional mimicry of a protein hormone by a peptide agonist: TheEPO receptor complex at 2.8."

Joliffe regards this EPO-mimicking molecule as "a significantmilestone," but only "an intermediate step." She observed that "manypeople in the field said this could never be done. It's a signalachievement in itself, but it's not the drug."

She and her team are now working "to reduce the peptide from 2,100molecular weight to 400 or 500." Their purpose is not to produce anorally administered EPO equivalent, but rather "to apply thistechnique to a variety of large protein cytokine molecules _ theinterleukins, the interferons, and others."

Protein engineer James Wells, who focuses on growth hormones atGenentech, of South San Francisco, wrote a commentaryaccompanying the two Science papers. He told BioWorld Today,"From a commercial point of view, that work provides a very stronglead candidate to consider further peptide reduction, and designing anorganic mimic of EPO, which could then be a pill _ with hugecommercial implications."

Amgen, of Thousand Oaks, Calif., said in mid-April of this year thatit has developed "a non-naturally occurring recombinant protein thathas the potential to become a new product to stimulate the productionof red blood cells." It anticipates clinical trials in 1997. n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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