Granted Patent B2

US 9,371,352 · App. 13/803,972 · Granted Jun 21, 2016

Abstract

Claims (75)

1. A modified ceramide-like glycolipid comprising a photoreactive group, wherein said photoreactive group has been covalently bound to the terminus of an acyl chain of the N-acyl lipophilic moiety of a ceramide-like glycolipid, wherein said photoreactive group is a benzophenone group, wherein said ceramide-like glycolipid is an α-galactosylceramide, and wherein said modified ceramide-like glycolipid is capable of binding to CD1d and enhancing the activity of natural killer T (NKT) cells.

2. The modified ceramide-like glycolipid of claim 1 , wherein said acyl chain is a linear acyl chain comprising at least 11 atoms, wherein the numbering of said atoms excludes hydrogens.

3. The modified ceramide-like glycolipid of claim 2 , wherein said acyl chain is selected from the group consisting of:

wherein Y is —O—, —CH 2 —, —S—, —OCH 2 —, —SCH 2 —, —CH 2 CH 2 —, or a bond; and PRG is the photoreactive group.

4. The modified ceramide-like glycolipid of claim 1 , wherein said α-galactosylceramide has the structure set forth in Formula II:

wherein

R1 is a linear or branched C 1 -C 27 alkane or C 2 -C 27 alkene; or R1 is —C(OH)—R3 wherein R3 is linear or branched C 1 -C 26 alkane or C 2 -C 26 alkene; and

R2 is one of the following (a)-(e):

(a) —CH 2 (CH 2 ) x CH 3 ,

(b) —CH(OH)(CH 2 ) x CH 3 ,

(c) —CH(OH)(CH 2 ) x CH(CH 3 ) 2 ,

(d) —CH═CH(CH 2 ) x CH 3 ,

(e) —CH(OH)(CH 2 ) x CH(CH 3 )CH 2 CH 3 ,

wherein X is an integer ranging from 4-17.

5. The modified ceramide-like glycolipid of claim 4 , wherein R2 is —CH(OH)—(CH 2 ) 13 CH 3 .

6. The modified ceramide-like glycolipid of claim 4 , wherein R1 is selected from the group consisting of (CH 2 ) 9 CH═CH—CH 2 —CH═CH(CH 2 ) 4 CH 3 , (CH 2 ) 8 CH═CH—CH 2 —CH═CH(CH 2 ) 4 CH 3 , (CH 2 ) 7 CH═CH—CH 2 —CH═CH(CH 2 ) 4 CH 3 , (CH 2 ) 3 CH═CH—CH 2 —CH═CH—CH 2 —CH═CH—CH 2 —CH═CH—(CH 2 ) 4 CH 3 , (CH 2 ) 3 CH═CH—CH 2 —CH═CH—CH 2 —CH═CH—CH 2 —CH═CH—CH 2 —CH═CH—CH 2 CH 3 , (CH 2 ) 7 CH═CH—CH 2 —CH═CH═(CH 2 ) 4 CH 3 , (CH 2 ) 7 CH═CH—CH═CH(CH 2 ) 5 CH 3 , (CH 2 ) 8 CH═CH—CH═CH(CH 2 ) 4 CH 3 , (CH 2 ) 9 CH═CH—CH═CH(CH 2 ) 5 CH 3 , (CH 2 ) 6 CH═CH—CH═CH—CH═CH(CH 2 ) 4 CH 3 , (CH 2 ) 6 CH═CH—CH═CH—CH═CH(CH 2 ) 4 CH 3 and (CH 2 ) 7 CH═CH—CH═CH—CH═CH(CH 2 ) 3 CH 3 .

7. The modified ceramide-like glycolipid of claim 1 , wherein said α-galactosylceramide has the structure set forth in Formula III:

wherein R is —C(O)R1, wherein R1 is a linear or branched C 1 -C 27 alkane or C 2 -C 27 alkene; or R1 is —C(OH)—R3 wherein R3 is a linear or branched C 1 -C 26 alkane or C 2 -C 26 alkene; or R1 is a C 6 -C 27 alkane or alkene wherein (i) the C 6 -C 27 alkane or alkene is substituted with a C 5 -C 15 cycloalkane, C 5 -C 15 cycloalkene, heterocycle, or aromatic ring or (ii) the C 6 -C 27 alkane or alkene includes, within the C 6 -C 27 alkyl or alkenyl chain, a C 5 -C 15 cycloalkane, C 5 -C 15 cycloalkene, heterocycle, or aromatic ring; or R1 is an optionally substituted aromatic ring, or an aralkyl, and

R2 is one of the following (a)-(e):

(a) —CH 2 (CH 2 ) x CH 3 ,

(b) —CH(OH)(CH 2 ) x CH 3 ,

(c) —CH(OH)(CH 2 ) x CH(CH 3 ) 2 ,

(d) —CH═CH(CH 2 ) x CH 3 ,

(e) —CH(OH)(CH 2 ) x CH(CH 3 )CH 2 CH 3 ,

wherein X is an integer ranging from 4-17.

8. The modified ceramide-like glycolipid of claim 7 , wherein R1 is substituted with oxo; hydroxy; halogen; phenyl; —OC(O)R6; —OR6; —C(O)R6; or N(R6) 2 , wherein each R6 is independently a C 1 -C 6 substituted alkyl, or a substituted aromatic ring optionally substituted with halogen; hydroxy; —OC(O)R7; —OR7; —C(O)R7 or N(R7) 2 , and

wherein each R7 is a substituted C 1 -C 6 alkyl.

9. The modified ceramide-like glycolipid of claim 7 , wherein R1 is selected from the group consisting of

where ( ) represent the point of attachment of R1 to the compound of Formula III.

10. The modified ceramide-like glycolipid of claim 1 , wherein said α-galactosylceramide is selected from the group consisting of: (2S,3S,4R)-1-O-(α-D-galactopyranosyl)-N-hexacosanoyl-2-amino-1,3,4-octadecanetriol (KRN7000), (2S,3S)-1-O-(α-D-galactopyranosyl)-N-hexacosanoyl-2-amino-1,3-octadecanediol), and (2S,3S,4R)-1-CH 2 -(α-galactopyranosyl)-N-hexacosanoyl-2-amino-1,3,4-octadecanetriol (α-C-GalCer).

11. The modified ceramide-like glycolipid of claim 1 , wherein said modified glycolipid has a structure selected from the group consisting of:

12. A method for synthesizing a modified glycolipid of Formula (VI) comprising a benzophenone group, said method comprising the steps of:

activating a carboxylic acid derivative of Formula (IV) to form an activated acyl derivative

and

coupling the activated acyl derivative with a glycolipid of Formula (V)

thereby forming the modified glycolipid comprising a benzophenone group of Formula (VI)

wherein

G is a linear or branched C 1 -C 27 alkane or C 2 -C 27 alkene; or G is —C(OH)—R3 wherein R3 is a linear or branched C 1 -C 26 alkane or C 2 -C 26 alkene; or R1 is a C 6 -C 27 alkane or alkene wherein (i) the C 6 -C 27 alkane or alkene is substituted with a C 5 -C 15 cycloalkane, C 5 -C 15 cycloalkene, heterocycle, or aromatic ring or (ii) the C 6 -C 27 alkane or alkene includes, within the C 6 -C 27 alkyl or alkenyl chain, a C 5 -C 15 cycloalkane, C 5 -C 15 cycloalkene, heterocycle, or aromatic ring; or R1 is an optionally substituted aromatic ring, or an aralkyl;

R 4 is an α-linked galactoside;

A is —O—, —CH 2 —, or is a single bond,

Y is —O—, —CH 2 — or —S—, and

R 2 is a substituted or an unsubstituted C 3 -C 40 alkyl, alkenyl or alkynyl chain; and

wherein said said modified glycolipid is capable of binding to CD1d and enhancing the activity of natural killer T (NKT) cells.

13. The method according to claim 12 , wherein R2 of the modified glycolipid comprising a benzophenone group of Formula (VI) is selected from the group consisting of:

(a) —CH 2 (CH 2 ) x CH 3 ,

(b) —CH(OH)(CH 2 ) x CH 3 ,

(c) —CH(OH)(CH 2 ) x CH(CH 3 ) 2 ,

(d) —CH═CH(CH 2 ) x CH 3 , and

(e) —CH(OH)(CH 2 ) x CH(CH 3 )CH 2 CH 3 , wherein x is an integer ranging from 0-40.

14. The method according to claim 12 , wherein:

G is —(CH 2 )n-, wherein n is an integer ranging from 0-20 and Y is O;

R2 is —CH(OH)(CH 2 ) x CH 3 , wherein x is an integer ranging from 4-17;

A is O; and

Y is O.

15. A modified glycolipid/protein complex comprising: a CD1d protein and the modified ceramide-like glycolipid of claim 1 , wherein said modified glycolipid is physically associated with said CD1d protein.

16. The modified glycolipid/protein complex of claim 15 , wherein said modified glycolipid/protein complex further comprises a β2-microglobulin physically associated with said CD1d.

17. The modified glycolipid/protein complex of claim 16 , wherein said modified glycolipid/protein complex enhances the activity of Natural Killer T (NKT) cells.

18. A modified glycolipid/protein complex comprising:

a) a CD1d protein;

b) a β2-microglobulin physically associated with said CD1d protein; and

c) a modified ceramide-like glycolipid;

wherein said modified ceramide-like glycolipid is covalently linked to said CD1d protein via photoactivation of a modified ceramide-like glycolipid of claim 1 , and wherein said modified glycolipid/protein complex enhances the activity of natural killer T (NKT) cells.

19. The modified glycolipid/protein complex of claim 18 , wherein said modified glycolipid/protein complex further comprises an antibody or fragment thereof specific for a target antigen, and wherein said antibody or antigen-binding fragment thereof is linked to said CD1d.

20. The modified glycolipid/protein complex of claim 19 , wherein said target antigen is a cell surface marker of tumor cells.

21. The modified glycolipid/protein complex of claim 20 , wherein said cell surface marker is selected from the group consisting of CEA, Her2/neu, EGFR type I or type II, CD19, CD20, CD22, Muc-1, PSMA, or STEAP.

22. The modified glycolipid/protein complex of claim 18 , further comprising an antigen.

23. The modified glycolipid/protein complex of claim 22 , wherein said antigen is an immunogenic polypeptide of a virus or a cell selected from the group consisting of a virus, bacterium, fungus, and tumor cell.

24. A composition comprising the modified glycolipid/protein complex of claim 18 , and a pharmaceutical carrier.

25. A method for producing a modified glycolipid/protein complex, said method comprising the steps of:

a) contacting the modified ceramide-like glycolipid of claim 1 with a CD1d protein; and

b) irradiating said modified glycolipid and said CD1d protein with ultraviolet light to produce a modified glycolipid/protein complex.

26. A method of inducing or enhancing an immune response against a tumor in a subject, comprising administering to said subject the modified glycolipid/protein complex of claim 20 .

27. The method of claim 26 , wherein said modified glycolipid/protein complex is administered in an amount sufficient to enhance the activity of Natural Killer T (NKT) cells in said subject.

28. The method of claim 26 , wherein said cell surface marker is selected from the group consisting of CEA, Her2/neu, EGFR type I or type II, CD19, CD20, CD22, Muc-1, PSMA, or STEAP.

29. A method for enhancing an immune response in a subject to a heterologous antigen, wherein said method comprises administering to said subject said heterologous antigen and the modified glycolipid/protein complex of claim 18 simultaneously, prior to, or subsequent to the administration of said heterologous antigen, and wherein said heterologous antigen comprises an immunogenic polypeptide.

Assignments (8)