Methods and compositions for generating bioactive assemblies of increased complexity and uses
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.
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), wherein the amino acid sequence of the AD moiety is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5; and (ii) a first effector protein selected from the group consisting of a first antibody and a first antigen-binding antibody fragment; 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 selected from the group consisting of a second antibody and a second antigen-binding antibody fragment;
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 first and second antibodies are selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.
3. The DNL complex of claim 1 , wherein the first and second antibodies or antibody fragments bind to a first antigen and a second antigens selected from the group consisting of carbonic anhydrase IX, alpha-fetoprotein, BrE3-antigen, 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, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG), HER2/neu, hypoxia inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA95, NCA90, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, a Thomson-Friedenreich antigen, a tumor necrosis antigen, VEGF, ED-B fibronectin, and 17-1A-antigen.
4. The DNL complex of claim 1 , wherein the first and second antibodies are selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), A20 (anti-CD20), L243 (anti-HLA class II), CC49 (anti-TAG-72), MN-14 (anti-CEA), MN-15 (anti-CEA), 679 (anti-HSG), L19 (anti-ED-B fibronectin), PAM4 (anti-MUC1), RS7 (anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab.