Conjugates of GM-CSF moiety and a polymer
Conjugates of a GM-CSF moiety and one or more water-soluble polymers are provided. Typically, the water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided are compositions comprising conjugates, methods of making conjugates, and methods of administering compositions comprising conjugates to a patient.
1 . A conjugate comprising the following structure:
wherein:
POLY is a water-soluble polymer;
(a) is either zero or one;
X 1 , when present, is a spacer moiety comprised of one or more atoms;
R 1 is an organic radical;
GM-CSF is a GM-CSF moiety.
2 . The conjugate of claim 1 , comprising the following structure:
wherein:
(n) is an integer having a value of from 3 to 4000;
X 1 is as previously defined;
R 1 is selected from the group consisting of methyl, ethyl, propyl, and isopropyl; and
GM-CSF is a human GM-CSF.
3 . The conjugate of claim 1 , comprising the following structure:
wherein (n) an integer having a value of from 3 to 4000 and GM-CSF is a human GM-CSF.
4 . A conjugate comprising the following structure:
wherein:
POLY is a water-soluble polymer;
(d) is either zero or one;
X 2 , when present, is a spacer moiety comprised of one or more atoms;
(b) is an integer having a value of one through ten;
(c) is an integer having a value of one through ten;
R 2 , in each occurrence, is independently H or an organic radical;
R 3 , in each occurrence, is independently H or an organic radical; and
GM-CSF is a GM-CSF moiety.
5 . The conjugate of claim 4 , comprising the following structure:
wherein:
(n) is an integer having a value of from 3 to 4000;
X 2 is as previously defined;
(b) is 2 through 6;
(c) is 2 through 6;
R 2 , in each occurrence, is independently H or lower alkyl; and
GM-CSF is a human GM-CSF.
6 . The conjugate of claim 5 , comprising the following structure:
wherein (n) is an integer having a value of from 3 to 4000, and GM-CSF is a human GM-CSF.
7 . The conjugate of claim 4 , comprising the following structure:
wherein:
each (n) is independently an integer having a value of from 3 to 4000;
X is as previously defined;
(b) is 2 through 6;
(c) is 2 through 6;
R 2 , in each occurrence, is independently H or lower alkyl; and
GM-CSF is a GM-CSF moiety.
8 . The conjugate of claim 5 , comprising the following structure:
wherein:
each (n) is independently an integer having a value of from 3 to 4000; and
GM-CSF is a human GM-CSF.
9 . A conjugate comprising a GM-CSF moiety comprising an internal amine covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a branched water-soluble polymer.
10 . The conjugate of claim 9 , wherein the conjugate further comprises an additional branched water-soluble polymer covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to the N-terminal amino acid residue.
11 . The conjugate of claim 9 , wherein the branched water-soluble polymer lacks a lysine residue in which polymers are connected to amine groups of the lysine residue via a —OCH 2 CONHCH 2 CO— linkage.
12 . The conjugate of claim 9 , wherein the branched water-soluble polymer comprises the following structure:
wherein each (n) is independently an integer having a value of from 3 to 4000.
13 . The conjugate of claim 1 , 4 or 9 , wherein the water-soluble polymer in each conjugate is selected from the group consisting of a poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).
14 . The conjugate of claim 13 , wherein each water-soluble polymer is a poly(alkylene oxide).
15 . The conjugate of claim 14 , wherein each poly(alkylene oxide) is a poly(ethylene glycol).
16 . The conjugate of claim 15 , wherein the poly(ethylene glycol) is terminally capped with an end-capping moiety selected from the group consisting hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.
17 . The conjugate of claim 15 , wherein the poly(ethylene glycol) is terminally capped with methoxy.
18 . The conjugate of claim 1 , 4 or 9 , wherein the water-soluble polymer has a total weight-average molecular weight in the range of from greater than 5,000 Daltons to about 150,000 Daltons.
19 . The conjugate of claim 18 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 6,000 Daltons to about 100,000 Daltons.
20 . The conjugate of claim 19 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 15,000 Daltons to about 85,000 Daltons.
21 . The conjugate of claim 20 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 20,000 Daltons to about 85,000 Daltons.
22 . The conjugate of claim 21 , wherein the poly(ethylene glycol has a total weight average molecular weight in the range of from about 20,000 Daltons to about 60,000 Daltons.
23 . The conjugate of claim 1 or 4 , wherein the water-soluble polymer is branched.
24 . The conjugate of claim 1 , 4 or 9 , wherein the GM-CSF moiety is selected from the group consisting of a human GM-CSF, and biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing.
25 . The conjugate of claim 1 , 4 or 9 , wherein the GM-CSF moiety is recombinantly derived.
26 . The conjugate of claim 1 , 4 or 9 , wherein not more than three water-soluble polymers are attached to the GM-CSF moiety.
27 . The conjugate of claim 1 , 4 or 9 in diPEGylated form.
28 . The conjugate of claim 1 , 4 or 9 in monoPEGylated form.
29 . The conjugate of claim 1 , 4 or 9 , wherein the GM-CSF moiety is nonglycosylated.
30 . The conjugate of claim 1 , 4 or 9 , wherein the GM-CSF moiety is glycosylated.
31 . A pharmaceutical composition comprising:
(i) a conjugate comprising a human GM-CSF covalently attached, either directly or through a spacer moiety comprised of one or more atoms, to a water-soluble polymer, wherein the water-soluble polymer has a weight-average molecular weight of greater than 5,000 Daltons; and
(ii) a pharmaceutically acceptable excipient,
wherein at least about 85% of the conjugates in the composition have from one to two polymers attached to the human GM-CSF.
32 . The composition of claim 31 , wherein each water-soluble polymer in each conjugate is selected from the group consisting of a poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).
33 . The composition of claim 32 , wherein each water-soluble polymer is a poly(alkylene oxide).
34 . The composition of claim 33 , wherein each poly(alkylene oxide) is a poly(ethylene glycol).
35 . The composition of claim 34 , wherein the poly(ethylene glycol) is terminally capped with an end-capping moiety selected from the group consisting hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.
36 . The composition of claim 34 , wherein the poly(ethylene glycol) is terminally capped with methoxy.
37 . The composition of claim 34 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from greater than 5,000 Daltons to about 150,000 Daltons.
38 . The composition of claim 37 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 6,000 Daltons to about 100,000 Daltons.
39 . The composition of claim 38 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 15,000 Daltons to about 85,000 Daltons.
40 . The composition of claim 39 , wherein the poly(ethylene glycol) has a total weight-average molecular weight in the range of from about 20,000 Daltons to about 85,000 Daltons.
41 . The composition of claim 40 , wherein the poly(ethylene glycol) has a total weight average molecular weight in the range of from about 20,000 Daltons to about 60,000 Daltons.
42 . The composition of claim 41 , wherein each water-soluble polymer in each conjugate is a linear water-soluble polymer.
43 . The composition of claim 31 , wherein each water-soluble polymer in each conjugate is a branched water-soluble polymer.
44 . The composition of claim 43 , wherein the branched water-soluble polymer lacks a lysine residue in which polymers are connected to amine groups of the lysine residue via a —CH 2 CONHCH 2 CO— group.
45 . The composition of claim 31 , with the proviso that when the water-soluble polymer is a branched water-soluble polymer, the branched water-soluble polymer lacks a lysine residue used to effect branching.
46 . The composition of claim 43 , wherein the branched water-soluble polymer comprises the following structure:
wherein each (n) is independently an integer having a value of from 3 to 4000.
47 . The composition of claim 31 , wherein the human GM-CSF comprises an amino acid sequence of SEQ ID NO: 1.
48 . The composition of claim 31 , wherein the human GM-CSF comprises an amino acid sequence of SEQ ID NO: 2.
49 . The composition of claim 31 , wherein the human GM-CSF is recombinantly derived.
50 . The composition of claim 48 , wherein the human GM-CSF is nonglycosylated.
51 . The composition of claim 48 , wherein the human GM-CSF is glycosylated.
52 . A pharmaceutical composition comprising: (i) the conjugate of claim 1 , 4 or 9 ; and (ii) a pharmaceutically acceptable excipient.
53 . The composition of claim 31 or 52 , wherein the composition is substantially free of albumin.
54 . The composition of claim 31 or 52 , wherein the composition is substantially free of proteins that do not have GM-CSF activity.
55 . The composition of claim 31 or 52 , wherein the composition is substantially free of noncovalently attached water-soluble polymers.
56 . The composition of claim 31 or 52 , in lyophilized form.
57 . The composition of claim 31 or 52 , in liquid form.
58 . The composition of claim 31 or 52 , wherein at least about 90% of the conjugates in the composition have from one to two polymers attached to the human GM-CSF.
59 . The composition of claim 58 , wherein at least about 95% of the conjugates in the composition have from one to two polymers attached to the human GM-CSF.
60 . A method for delivering a conjugate to a patient, the method comprising the step of administering to the patient a pharmaceutical composition of claim 31 or 52 .
61 . The method of claim 60 , carried out by subcutaneous injection.
62 . A method for making a conjugate comprising contacting, under conjugation conditions, a GM-CSF moiety with a polymeric reagent to result in a conjugate of claim 1 , 4 or 9 .
63 . The method of claim 62 , wherein protecting and deprotecting steps are not performed.