Novel Method Could Improve the Performance of Proteins Used Therapeutically

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http://www.sciencedaily.com/releases/2011/03/110309125157.htm

Novel Method Could Improve the Performance of Proteins Used Therapeutically

ScienceDaily (Mar. 9, 2011) — Whitehead Institute scientists have created a

method that site-specifically modifies proteins to exert control over their

properties when administered therapeutically. The technique should be useful to

increase potency, slow metabolism, and improve thermal stability of

therapeutically useful proteins, such as interferon alpha 2 (IFN-alpha 2), which

is used to treat variety of diseases, including leukemia, melanoma, and chronic

hepatitis C.

The method, reported this month in Proceedings of the National Academy of

Sciences (PNAS), uses the enzyme sortase A and can be applied to tailor proteins

that possess a structure found in IFN-alpha 2, referred to as a four-helix

bundle. Such proteins include erythropoietin (EPO), granulocyte

colony-stimulating factor 3 (GCSF-3, known as filgrastim and marketed as

Neupogen®), interleukin (IL) 2 (known as aldesleukin and marketed as

Proleukin®), IL-4, IL-7, IL9-, and IL-15.

" In the course of this work, the first author of the PNAS paper, Maximilian

Popp, together with other members of the lab, has put together a nice palette of

sortase-based techniques that now allow us to modify a large variety of

different proteins, and equip them with properties and behaviors that cannot be

easily specified by more standard molecular biological techniques, " says

Whitehead Member Hidde Ploegh. " I see the value of these approaches first and

foremost in their general applicability and ease of use. "

IFN-alpha 2 is a cytokine, a hormone-like substance that usually acts on cells

other than those that produce the protein. Upon binding the cytokine, the

recipient cell responds, for example by starting to divide and proliferate, or

by exercising certain functions of benefit to the organism. Like other cytokines

used for therapeutic purposes, IFN-alpha 2 can be a finicky drug. It is

thermally unstable and must be continuously refrigerated to maintain its

potency, a requirement that limits IFN-alpha 2's use in areas with intermittent

or no electricity. Also, IFN-alpha 2's relatively short half-life (and resulting

rapid clearance from the body) often necessitates frequent injections when the

drug is used to treat certain conditions.

To keep therapeutic IFN-alpha 2 active in the body longer, the current strategy

is to tack long polyethylene glycol (PEG) chains onto the protein to turn them

into effective drugs. This so-called PEGylation not only masks IFN-alpha 2 from

the patient's immune system but also increases the time the body needs to break

it down. However, because current approaches to PEGylation are not specific, the

PEG chains can block or alter the protein's normal binding site -- an unintended

consequence of this modification that can diminish IFN-alpha 2's potency by as

much as 90%.

Seeking greater precision in attaching PEG chains, Popp, who is a graduate

student in the Ploegh lab, used the enzyme sortase A to cleave IFN-alpha 2 at a

specific site on the protein, engineered so that it would be recognized by the

sortase. Then, a small molecule bearing the PEG chain was attached at the site

cleaved by sortase. When Popp tested for biological activity, the resulting

IFN-alpha 2 was highly potent, indicating that the PEG chains were not

interfering with the drug's binding ability.

Popp also used sortase A to suture PEG chains to the cytokine GCSF-3. When he

tested the PEGylated version in mice, it remained in the bloodstream

significantly longer and evoked a more robust and prolonged response than a

non-PEGylated version. By using sortase A's inherent precision to attach PEG

chains, Popp could replace the less precise chemistry-based technique with a

highly effective method that should have broader applications.

Next, Popp addressed IFN-alpha 2's thermal stability. Previously, the Ploegh lab

stabilized linear polypeptides like IFN-alpha 2 by molecularly gluing their ends

together to form circles. A few such cyclic proteins are found in nature. Once

circularized, cyclic proteins are often more stable than their linear

precursors. This forced looping can interfere with the function of some

cell-signaling proteins, but because IFN-alpha 2's binding site is not near its

ends, the function of IFN-alpha 2 is unaffected when its ends are joined to form

a circle.

To create a cyclic version of IFN-alpha 2, Popp used sortase A to join the two

ends of IFN-alpha 2. When he heated the cyclic form of IFN-alpha 2, it was more

resistant to breakdown than its linear counterpart and remained biologically

potent even after boiling. Popp then tested the circular, PEGylated version and

the linear version in mice. The modified version was metabolized more slowly

than the linear version and maintained its thermal stability, demonstrating that

this simple technique can significantly enhance desirable properties of a

therapeutically relevant protein without sacrificing its potency.

" We really take advantage of the site specificity of the sortase enzyme. Placing

a molecular suture like that can't really be done by other means. So I think

this method is of value to the protein engineering field in general, " says Popp.

" The reaction itself is easy, but it took some time to actually figure out how

to do these transformations. Once we figured that out, the technique was robust

and reproducible. "

This research was supported by the National Institutes of Health (NIH).

1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge,

MA 02142 2. Department of Biology, Massachusetts Institute of Technology, 77

Massachusetts Avenue, Cambridge, MA 02142