Methods and Compositions for Generating Bioactive Assemblies of Increased Complexity and Uses

Abstract

The present invention concerns methods and compositions for making and using bioactive assemblies of defined compositions, which may have multiple functionalities and/or binding specificities. In particular embodiments, the bioactive assembly is formed using dock-and-lock (DNL) methodology, which takes advantage of the specific binding interaction between dimerization and docking domains (DDD) and anchoring domains (AD) to form the assembly. In various embodiments, one or more effectors may be attached to a DDD or AD sequence. Complementary AD or DDD sequences may be attached to an adaptor module that forms the core of the bioactive assembly, allowing formation of the assembly through the specific DDD/AD binding interactions. Such assemblies may be attached to a wide variety of effector moieties for treatment, detection and/or diagnosis of a disease, pathogen infection or other medical or veterinary condition.

What is claimed is: 
     
         1 . A dock and lock (DNL) complex comprising:
 a) a first fusion protein comprising (i) an anchoring domain (AD) from an A-kinase anchoring protein (AKAP; and (ii) a first effector protein that is an antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof binds to a tumor associated antigen (TAA); and   b) a second fusion protein comprising (iii) a dimerization and docking domain (DDD) moiety, wherein the amino acid sequence of the DDD moiety is selected from the group consisting of residues 1-44 of human protein kinase A (PKA) RIIα, residues 1-44 of human PKA RIIβ and residues 12-61 of human PKA RIα; and (iv) a second effector protein that is a toxin, wherein the toxin is selected from the group consisting of ricin, abrin, ribonuclease, ranpirnase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,  Pseudomonas  exotoxin and  Pseudomonas  endotoxin;   
       wherein two copies of the DDD moiety form a dimer that binds to the AD moiety to form the DNL complex. 
     
     
         2 . The DNL complex of  claim 1 , wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a human antibody. 
     
     
         3 . The DNL complex of  claim 1 , wherein the antibody fragment is selected from the group consisting of a Fab fragment, a F(ab) 2  fragment, a Fab′ fragment, a F(ab′) 2  fragment, a single domain (DAB) antibody fragment, a scFv, a diabody and a triabody. 
     
     
         4 . The DNL complex of  claim 1 , wherein the TAA is selected from the group consisting of carbonic anhydrase IX, alpha-fetoprotein, CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, EGFR, EGP-1, EGP-2, Flt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG), HER2/neu, hypoxia inducible factor (HIF-1), IL-2, IL-6, IL-8, insulin-like growth factor-1 (IGF-1), KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA95, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, TAG-72, tenascin, TRAIL receptors, Tn antigen, a tumor necrosis antigen, VEGF, and ED-B fibronectin. 
     
     
         5 . The DNL complex of  claim 4 , wherein the antibody is selected from the group consisting of hLL1 (anti-CD74), hLL2 (anti-CD22), hA20 (anti-CD20), L243 (anti-HLA class II), hCC49 (anti-TAG-72), hMN-14 (anti-CEA), hMN-15 (anti-CEA), h679 (anti-HSG), L19 (anti-ED-B fibronectin), hPAM4 (anti-mucin), hRS7 (anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab.