Site Matters: A Case Study

Ambrx selected human growth hormone (hGH) to demonstrate the utility of protein medicinal chemistry via its ReCODE™ protein engineering platform. Growth hormone represents a significant, expanding market: worldwide sales are in excess of $2.5 billion and growing. Ambrx’s hGH represents a best in class product opportunity with optimized pharmacology and preserved potency. The goal of this project is to deliver a product with a once-weekly dosing frequency for the treatment of adults and children with growth hormone deficiency. Ambrx has additional projects within the areas of metabolic diseases, CNS disorders, asthma, and infectious diseases. Approaches in these projects include both therapeutic proteins and antibodies.

The grounding hypothesis for using ReCODE™ to enhance hGH performance centered on two objectives:

1. Site-directed placement of a chemically reactive amino acid to provide a single point of attachment for a polyethylene glycol (PEG) polymer through orthogonal chemistry that does not react with any of the 20 natural amino acids
2. Optimize placement of the PEGylation site on the surface of the hGH protein to improve longevity of action while maintaining potency

The process for engineering hGH began with incorporation of a novel amino acid, para-acetyl-phenylalanine at sites specified by the amber codon. Para-acetyl-phenylalanine (pAcF) is a derivative of phenylalanine that has been modified to include a ketone functional group. The ketone is chemically inert but it reacts efficiently and selectively with a specially-derivatized PEG polymer. As a result, Ambrx can introduce a single, selective attachment point at any site on the protein’s surface, enabling protein engineering in a manner not previously possible.

Through supplementation of pAcF into the growth media of E. coli reconstituted with plasmids that direct the synthesis of tRNAo and tRNAo synthetase, pAcF is incorporated into recombinant hGH molecules at different single sites (depicted in red in the composite below).

A key feature of pAcF is its selectivity of chemical reaction. In the figure below, reaction of a specially-derivatized 5 kDa PEG polymer shifts the molecular size of hGH substituted with pAcF (incorporated at position 134) but the PEG does not modify unsubstituted hGH. This selectivity of reaction contrasts with succinimidyl-activated PEG polymers that react with multiple surface lysines, creating a heterogeneous mixture of products that differ in the number of PEG’s added and in the sites at which they are attached. Importantly, many lysine residues are present at key sites; if these residues are disrupted through non-specific conjugation, potency or safety of the molecule may be compromised.

The site of PEGylation has significant impact on pharmacology and efficacy. In this and multiple other projects Ambrx has demonstrated that “site matters”. Molecules that vary only in the location of the site of attachment can exhibit significant differences in pharmacology, receptor binding, efficacy and biophysical stability. Ambrx designated a lead in the PEGylated hGH project based upon a rigorous analysis of these parameters. In February 2007, Ambrx advanced ARX-201, the lead from this program into phase I/II clinical trials. The results from this multi-dose pharmacokinetic and pharmacodynamic trial demonstrate a sustained pharmacodynamic response and excellent safety profile. This program continues to advance under a strategic collaboration with EMD Serono announced in June 2007.