Carbohydrate-directed cross-linking reagents

• US 5,929,211 A
• Filed: 09/04/1997
• Issued: 07/27/1999
• Est. Priority Date: 08/05/1992
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Ashkenazi, Avi J., Chamow, Steven M., Kogan, Timothy P.
• Primary Examiner(s): McKane, Joseph K.
• Application Number:
  US08/923,590
• Time in Patent Office: 691 Days
• Field of Search: 548/546, 548/544, 548/547, 548/548, 548/549, 530/351, 530/359, 530/387.1, 530/395, 530/396, 530/397, 530/398, 530/399, 530/380, 530/389.1, 530/389.2, 530/391.1, 530/391.3, 530/391.5, 530/391.7 , 530/391.9, 514/8, 514/12, 514/21, 424/85.1, 424/178.1, 424/179.1, 424/194.1, 424/195.11
• US Class Current: 530/351
• CPC Class Codes: C07D 207/452 with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: Y10S 435/974 Aids related test
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier

Abstract

The invention provides heterobifunctional cross-linking reagents and methods of using the cross-linking reagents. The cross-linking reagents of the invention combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling of aldehydes to free thiols. In the methods of the invention, human immunodeficiency virus (HIV) infected cells can be detected using conjugates that include CD4 molecules conjugated to detectable markers via the disclosed cross-linking reagents.

Claims

1. A conjugate molecule having a glycoprotein covalently attached to a thiol-containing compound via a compound of formula I ##STR3## wherein the conjugate has a hydrazide moiety and a maleimide moiety, wherein the hydrazide and the maleimide moieties are separated by a spacer X,

wherein X is an aliphatic hydrocarbon spacer of 6 to about 25 carbon atoms or consists of an aliphatic hydrocarbon chain and an alicyclic or aromatic moiety having a total number of 6 to about 25 carbon atoms,

wherein X is covalently attached to the hydrazide moiety and X is covalently attached to the heteronitrogen of the maleimide moiety,

wherein the glycoprotein is covalently attached to the hydrazide via a hydrazone linkage;

and

wherein the thiol-containing compound is covalently attached to a heterocyclic carbon atom of the maleimide via a thioether linkage.

2. The conjugate of claim 1 wherein said thiol-containing compound is a toxin.

3. The conjugate of claim 1 wherein said thiol-containing compound is a component of a liposome and said liposome contains a toxin.

4. The conjugate of claim 1 wherein said glycoprotein is a cell surface receptor.

5. The conjugate of claim 4 wherein said cell surface receptor is a cytokine, a hormone or a growth factor receptor.

6. The conjugate of claim 4 wherein said cell surface receptor is a member of the immunoglobulin gene superfamily.

7. The conjugate of claim 6 wherein said cell surface receptor is CD4.

8. The conjugate of claim 7 wherein said thiol-containing compound consists of an anti-human immunodeficiency virus agent.

9. The conjugate of claim 7 wherein said thiol-containing compound is a component of a liposome and said liposome contains a toxin, and wherein said toxin is an anti-human immunodeficiency virus agent.

10. The conjugate of claim 7 wherein said thiol-containing compound consists of a detectable marker.

11. The conjugate of claim 1 wherein in said compound of formula I the aliphatic hydrocarbon chain has 2 to about 10 carbon atoms.

12. The conjugate of claim 1 wherein in said compound of formula I the alicyclic or aromatic moiety is linked to the maleimido terminus.

13. The conjugate of claim 1 wherein in said compound of formula I the aromatic moiety is monocyclic.

14. The conjugate of claim 1 wherein said compound of formula I is 4-(4-N-maleimidophenyl)butyric acid anhydride.

15. The conjugate of claim 10 wherein said detectable marker is fluorescein.

16. The conjugate of claim 8 wherein said anti-human immunodeficiency virus agent is AZT.

17. The conjugate of claim 8 wherein said anti-human immunodeficiency virus agent is an anti-CD3 or an anti-CD16 antibody.