Anti-Alpha V Immunoliposome Composition, Methods, and Uses

Abstract

An immunoliposome composition targeted to the alphaV-integrin subunit of integrin receptors comprised of ligand-targeted liposomes bearing at least one targeting-ligand derived from an antibody and having binding specificity for at least one integrin receptor comprising an alpha V subunit including αvβ1, αvβ3 αvβ5, αvβ6, or αvβ8 integrin cell receptors is described. The antibody-derived targeting ligand may be a Fab′ fragment, a scFv, or the like. Binding of the immunoliposome to αv-integrin expressing cells, preferably results in internalization of the immunoliposome for cytoplasmic delivery of a liposome-entrapped agent.

1 . An antibody derived targeting-ligand capable of monovalent binding and having specificity for alpha V integrin comprising:
 (a) a first polypeptide domain comprising the light chain variable region of a parent monoclonal antibody having specificity for alpha V-integrin or a conservative modification or fragment thereof that retains the binding specificity;   (b) a second polypeptide domain comprising the heavy chain variable region of a parent monoclonal antibody having specificity for alpha V-integrin or a conservative modification or fragment thereof that retains the binding specificity; and wherein the targeting ligand retains specificity for at least one alphaV integrin.   
     
     
         2 . The antibody derived targeting-ligand of  claim 1 , wherein the light chain variable domain comprises residues 1-108 of SEQ ID NO: 2 and the heavy chain variable domain comprises residues 1-119 of SEQ ID NO: 1. 
     
     
         3 . The antibody derived targeting-ligand of  claim 1 , further comprising at least one peptide linker linking said first and second polypeptides (a) and (b) into a single chain Fv polypeptide having binding affinity specificity for alpha V integrin. 
     
     
         4 . The single chain Fv polypeptide of  claim 3  wherein the linker has an amino acid sequence of the formula (Gly 4 Ser) n  where n is an integer of 1-4. 
     
     
         5 . The single chain Fv polypeptide of  claim 4  where n is 3. 
     
     
         6 . The single chain Fv polypeptide of  claim 5  wherein the heavy chain variable region has a leucine substitution at the 18 th  amino acid of SEQ ID NO: 1. 
     
     
         7 . The single chain Fv polypeptide of  claim 3 , which competes with the monoclonal antibody CNTO95 for binding to at least one human alpha V integrin subunit. 
     
     
         8 . The antibody derived targeting-ligand of  claim 1 , comprising a Fab fragment wherein the said first and second polypeptides (a) and (b) comprise polypeptides linked by a disulfide bond. 
     
     
         9 . The Fab fragment of  claim 8 , produced by an enzymatic digestion of the monoclonal antibody CNTO95. 
     
     
         10 . The Fab fragment of  claim 9 , wherein the enzymatic digestion step uses pepsin. 
     
     
         11 . The Fab fragment of  claim 9 , wherein the enzymatic digestion step uses papain. 
     
     
         12 . The Fab fragment of  claim 8 , wherein the Fab is secreted from a host cell line. 
     
     
         13 . The Fab fragment of  claim 12 , wherein the host cell line is transfected with a nucleic acid coding for the amino acid sequence of SEQ ID NO: 3. 
     
     
         14 . The antibody derived targeting-ligand of any of  claims 1 - 4 , wherein the targeting-ligand comprises a predetermined site for chemical conjugation. 
     
     
         15 . An isolated nucleic acid molecule encoding the antibody derived targeting-ligand of  claim 1 . 
     
     
         16 . An isolated nucleic acid encoding a single chain Fv polypeptide of  claim 1  comprising a nucleic acid sequence according to SEQ ID NO: 5. 
     
     
         17 . The isolated nucleic acid molecule of  claim 15  wherein the nucleic acid molecule is incorporated into an expression vector. 
     
     
         18 . An isolated nucleic acid vector comprising an isolated nucleic acid according to  claim 15 . 
     
     
         19 . A prokaryotic or eukaryotic host cell comprising an isolated nucleic acid according to  claims 15 ,  16 ,  17 , or  18 . 
     
     
         20 . A host cell according to  claim 19 , wherein said host cell is at least one selected from  E. coli , COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma cells, Perc.6, or any derivative, immortalized or transformed cell thereof. 
     
     
         21 . A pharmaceutical composition comprising the antibody derived targeting-ligand of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         22 . A pharmaceutical composition comprising the antibody derived targeting-ligand of  claim 1  covalently linked to, or conjugated to a non-biologic polymer, which polymer optionally is further covalently linked to a lipid, an active, or a reporter molecule. 
     
     
         23 . A pharmaceutical composition comprising the antibody derived targeting-ligand of  claim 22 , in which the biological polymer conjugate is inserted into the surface of a liposome. 
     
     
         24 . The composition according to  claim 23 , wherein the liposome has an anti-neoplastic agent encapsulated within. 
     
     
         25 . A composition according to  claim 24 , wherein the anti-neoplastic agent is selected from a radiopharmaceutical, an estrogen receptor modulator, a retinoid, a topoisomererase inhibitor, a cytotoxin, an alkylating agent, a nitrogen mustard, a nitrosourea, an antimetabolite, a mitotic inhibitor, and a radiosensitizer. 
     
     
         26 . A composition according to  claim 25 , wherein the anti-neoplastic agent is doxorubicin. 
     
     
         27 . An isolated nucleic acid molecule encoding the single chain Fv polypeptide of  claim 1 . 
     
     
         28 . A method for producing the single chain Fv polypeptide of  claim 1 , comprising translating a nucleic acid according to  claims 15 - 18  under conditions in vitro, in vivo or in situ, such that the antibody derived targeting-ligand of  claim 1  is expressed in detectable or recoverable amounts. 
     
     
         29 . A method of inhibiting growth of a cell expressing alpha v integrin, comprising contacting the cell with an effective amount of a pharmaceutical composition according to  claims 21 - 26  such that the growth of the cell is inhibited.