Novel bioactive lipid derivatives, and methods of making and using the same

Abstract

Compositions and methods for producing monoclonal antibodies and their derivatives reactive against bioactive lipid targets are described. These compositions include derivatized lipids, each of which comprises a bioactive lipid that having a polar head group and at least one hydrocarbon chain (e.g., a lysolipid such as lysophosphatidic acid or sphingosine-1-phosphate) in which a carbon atom has been derivatized with a pendant reactive group; immunogens made by linking a derivatized lipid to a carrier moiety (e.g., a carrier protein, polyethylene glycol, colloidal gold, alginate, or a silicone bead); monoclonal antibodies and derivatives produced by immunizing an animal with such an immunogen; and therapeutic and diagnostic compositions containing such antibodies and antibody derivatives. Methods for making such derivatized lipids, immunogens, and monoclonal antibodies and derivatives, methods for detecting such antibodies once generated, and therapeutic and diagnostic methods for using such antibodies and derivatives, are also described.

We claim: 
     
         1 . A method, comprising:
 a. immunizing an immune competent non-human mammalian host with an amount of a derivatized bioactive lipid conjugate sufficient to elicit antibody production in the immune competent non-human mammalian host to the bioactive lipid component of derivatized bioactive lipid conjugate, wherein the derivatized bioactive lipid conjugate comprises a derivatized bioactive lipid that comprises a bioactive lipid having a head group and at least one hydrocarbon chain covalently linked to the head group, wherein a carbon atom within the hydrocarbon chain is derivatized with an optionally protected pendant reactive group to form the derivatized bioactive lipid, and a carrier moiety conjugated to the derivatized bioactive lipid via the pendant reactive group; and   b. identifying antibody-producing cells isolated from the immune competent non-human mammalian host that produce antibody specifically reactive with the bioactive lipid of the derivatized bioactive lipid conjugate, wherein the identification comprises using a support, optionally a solid support, modified with the derivatized bioactive lipid to detect cells that produce antibody specifically reactive with the bioactive lipid.   
     
     
         2 . A method according to  claim 1  further comprising producing an antibody-producing cell line from the antibody-producing cells. 
     
     
         3 . A method according to  claim 2  further comprising producing a derivative of the antibody specifically reactive with the bioactive lipid of the derivatized bioactive lipid conjugate, which derivative is selected from the group consisting of a humanized antibody and a chimeric antibody. 
     
     
         4 . A method according to  claim 3  further comprising isolating the derivative of the antibody specifically reactive with the bioactive lipid of the derivatized bioactive lipid conjugate. 
     
     
         5 . A method according to  claim 1  wherein the bioactive lipid is a lysolipid, optionally a lysolipid selected from the group consisting of a sphingolipid and a sphingolipid metabolite, optionally selected from the group consisting of ceramide, ceramide-1-phosphate, N-acetyl-ceramide-1-phosphate, lysophosphatidic acid, sphingosine-1-phosphate, sphingosine, sphingosylphosphorylcholine, dihydrosphingosine, and dihydrosphingosine-1-phosphate. 
     
     
         6 . A method according to  claim 1  wherein the carbon chain of the bioactive lipid is derivatized with a protected pendant reactive group. 
     
     
         7 . A method according to  claim 6  wherein the pendant reactive group is selected from the group consisting of a sulfhydryl group, a carboxylic acid group, a cyano group, an ester, a hydroxy group, an alkene, an alkyne, an acid chloride group, and a halogen atom. 
     
     
         8 . A method according to  claim 1  wherein the carrier moiety is selected from the group consisting of a carrier protein (optionally selected from the group consisting of a keyhole limpet hemocyanin and albumin), polyethylene glycol, colloidal gold, an adjuvant, and a silicone bead.