Amunix, Inc.

Half-life Extension Technology (XTEN)

Half life extension is a critical aspect of developing a successful protein pharmaceutical as not all efficacious payloads have natural half lives that are amenable to reasonable dosing intervals. One common approach is chemical attachment of a polyethylene glycol (PEG) moiety that bulks the product and reduces kidney clearance. PEGylation can also reduce payload immunogenicity and increase payload stability by steric shielding. There are, however, several manufacturing disadvantages and safety concerns with PEGylation.

The most notable limitation of PEGylation is the requirement for a chemical conjugation step. This extra conjugation step significantly increases production cost and frequently yields complex product mixtures that include many inactive variants.

Although site-specific conjugation strategies have been developed that improve the yield and specificity of the conjugation site for some products, issues remain with the cost and intrinsic heterogeneity of the PEG itself. Our consultants estimate that the XTEN process reduces the cost of goods by almost 10-fold relative to PEGylation, depending on the details.

PEGylated proteins have in some cases been observed to cause renal tubular vacuolation. PEG has historically been considered to be non-immunogenic, but immune responses to PEG have been reported. A recent increase in pre-existing antibodies to PEG has been attributed to the growing use of PEG in cosmetic products.

An alternative to PEG would ideally confer similar serum half-life in humans, yet be composed of a non-immunogenic, homogeneous, and biodegradable polymer. A simple polypeptide chain is capable of forming an extended conformation similar to PEG. A polypeptide chain also has the advantage of being produced recombinantly fused to the protein drug of interest without requiring an independent conjugation step during manufacture.

Due to XTEN’s extended structure, it provides a bulking effects similar to PEG, with associated improvements in payload immunogenicity and stabilization. In addition, standard molecular biology techniques allow precise control of the side chain composition of any XTEN sequence, allowing optimization of immunogenicity and manufacturing properties.

For example, the addition of XTEN to Exenatide, a 39AA peptide sold as Byetta by Amylin Inc. for diabetes treatment, mediates five valuable advantages. First, because of much flatter PK profile, the dosing can be changed from twice a day to as little as once a month. Exenatide causes a high frequency of nausea and vomiting, which is attributed to the rapid, sharp concentration changes. The much flatter PK profile of the XTEN product is expected to greatly decrease these severe side effects. Exenatide is made by a complex synthetic chemical process, but the manufacturer would prefer a recombinant process, as it can be run at a larger scale. Most peptides, including Exenatide, are hydrophobic and difficult to formulate.

Instead, the XTEN fusion product is very hydrophilic and can be concentrated to >60mg/ml. Exenatides causes an immune response in 60% of patients, whereas the XTEN fusion is expected to be less immunogenic, based on our own work and many other studies demonstrating the reduction of immunogenicity upon addition of XTEN or PEG.

We have extensively studied the immunogenicity of XTENs in animals. We immunized 4 species of mammals (mice, rats, rabbits and cynomolgus monkeys) with 10 XTENs representing 9 different average compositions. All were composed of different combinations of amino acids G, E, S, T, A and P and comprise a total of 436 different 9-mer peptides. We used 3-10 subcutaneous injections. None of the XTENs yielded a significant immune response in any of the animals, the highest signal being 0.30 OD at a 1:100 dilution (see slides). Even with complete Freund’s adjuvant and multiple exposures, we have repeatedly failed to obtain antisera to these proteins in mice and rabbits. In four separate attempts, panning of antibody phage display libraries by Morphosys has failed to obtain antibodies that bind XTEN.