POLY-PEGYLATED PROTEASE INHIBITORS

1 . A compound comprising

(i) a Kunitz domain polypeptide that comprises a Kunitz domain that binds to and inhibits a protease, wherein the polypeptide comprises the DX-88 amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence that differs by at least one, but fewer than six amino acids from the DX-88 amino acid sequence set forth in SEQ ID NO:24; and

(ii) a plurality of polyethylene glycol moieties attached to the Kunitz domain polypeptide, wherein the average molecular weight of each of the moieties is less than 12 kDa, and each accessible primary amine of the Kunitz domain polypeptide is attached to one of the moieties.

2 . The compound of claim 1 wherein the average molecular weight of each of the moieties is less than 8 kDa.

3 . The compound of claim 1 wherein each moiety has a molecular weight between 3-8 kDa.

4 . The compound of claim 1 wherein the Kunitz domain polypeptide has a molecular weight less than 8 kDa, and the compound has a molecular weight greater than 16 kDa.

5 . The compound of claim 1 wherein the plurality of polyethylene glycol moieties consists of four or five moieties, each attached to a different accessible primary amine.

6 . The compound of claim 1 wherein each lysine is coupled to one of the moieties of the plurality.

7 . The compound of claim 6 wherein the Kunitz domain polypeptide comprises an N-terminal primary amine, and each lysine and the N-terminal primary amine is coupled to one of the moieties.

8 . The compound of claim 1 wherein the Kunitz domain polypeptide does not include a lysine in the Kunitz domain binding loops.

9 . The compound of claim 1 wherein the Kunitz domain polypeptide includes at least two lysines in the framework region of the Kunitz domain.

10 . The compound of claim 1 wherein the Kunitz domain polypeptide comprises three lysines in the framework region of the Kunitz domain.

11 . The compound of claim 10 wherein the Kunitz domain polypeptide comprises four lysines in the framework region of the Kunitz domain.

12 . The compound of claim 1 wherein the Kunitz domain polypeptide comprises a framework region that is identical to a corresponding region of a human Kunitz domain.

13 . The compound of claim 12 wherein the Kunitz domain polypeptide comprises a framework region that is identical to corresponding residues in a LACI Kunitz domain or an ITI Kunitz domain.

14 - 22 . (canceled)

23 . A preparation that comprises Kunitz domain polypeptides that specifically bind and inhibit a protease, wherein at least 80% of the Kunitz domain polypeptides in the preparation (i) bind and inhibit the protease, wherein the at least 80% of the Kunitz domain polypeptides comprise the DX-88 amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence that differs by at least one, but fewer than six amino acids from the DX-88 amino acid sequence set forth in SEQ ID NO:24; and

(ii) have a polyethylene glycol moiety attached at a first common site and a polyethylene glycol moiety attached at a second common site and wherein the average molecular weight of each of the attached polyethylene glycol moieties is less than 12 kDa.

24 . The preparation of claim 23 wherein the average molecular weight of each of the attached polyethylene glycol moieties is less than 10 kDa.

25 - 52 . (canceled)

53 . A preparation that comprises Kunitz domain polypeptides that comprise the amino acid sequence of DX-88, wherein at least 80% of the DX-88-containing Kunitz domain polypeptides in the preparation have a polyethylene glycol moiety attached to each of three lysine residues and to the N-terminus of the polypeptide.

54 . (canceled)

55 . A method of providing a pegylated Kunitz domain, the method comprising:

providing a polypeptide that comprises the DX-88 sequence set forth in SEQ ID NO:24 or an amino acid sequence that differs by at least one, but fewer than six amino acids from the DX-88 sequence set forth in SEQ ID NO:24; and

contacting the polypeptide with activated polyethylene glycol, of average molecular weight less than 12 kDa, under conditions in which a plurality of polyethylene glycol moieties are attached to the polypeptide, at least one of which is attached to the lysine and at least one is attached to the N-terminal primary amine.

56 - 67 . (canceled)

68 . A method of treating a disorder characterized by excessive or undesired activity of a protease, the method comprising, administering to a subject having the disorder or suspected of having the disorder to pharmaceutical composition comprising the preparation of claim 1 , wherein the Kunitz domain polypeptide of the preparation inhibits the protease.

69 - 74 . (canceled)

75 . A preparation comprising molecules that comprise:

(i) a Kunitz domain polypeptide that comprises a Kunitz domain that binds to and inhibits a protease, wherein the polypeptide comprises the DX-88 amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence that differs by at least one, but fewer than six amino acids from the DX-88 amino acid sequence set forth in SEQ ID NO:24; and

(ii) a plurality of polyethylene glycol moieties attached to the Kunitz domain polypeptide, wherein the average molecular weight of each of the moieties is less than 12 kDa.

76 - 78 . (canceled)

79 . A method of treating a disorder characterized by excessive or undesired activity of a protease, the method comprising, administering to a subject having the disorder or suspected of having the disorder to pharmaceutical composition comprising the preparation of claim 75 , wherein the Kunitz domain polypeptide of the preparation inhibits the protease.