Multivalent Immunoglobulin-Based Bioactive Assemblies

Abstract

The present invention concerns methods and compositions for stably tethered structures of defined compositions, which may have multiple functionalities and/or binding specificities. Preferred embodiments concern hexameric stably tethered structures comprising one or more IgG antibody fragments and which may be monospecific or bispecific. The disclosed methods and compositions provide a facile and general way to obtain stably tethered structures of virtually any functionality and/or binding specificity. The stably tethered structures may be administered to subjects for diagnostic and/or therapeutic use, for example for treatment of cancer or autoimmune disease. The stably tethered structures may bind to and/or be conjugated to a variety of known effectors, such as drugs, enzymes, radionuclides, therapeutic agents and/or diagnostic agents.

1 . A fusion protein comprising: (i) an antibody, an antigen-binding antibody fragment or a cytokine conjugated to (ii) a dimerization and docking domain (DDD) moiety of human protein kinase A regulatory subunit RIα, RIβ, RIIα and RIIα. 
     
     
         2 . The fusion protein of  claim 1 , wherein the amino acid sequence of the DDD moiety is selected from the group consisting of residues 1 to 44 of human PKA RIIα, residues 1 to 44 of human PKA RIIβ, residues 12 to 61 of human PKA RIα and residues 13 to 66 of human PKA RIβ. 
     
     
         3 . The fusion protein of  claim 1 , wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), A20 (anti-CD 20 ), L243 (anti-HLA class II), CC49 (anti-TAG-72), MN-14 (anti-CEA), MN-15 (anti-CEA), 679 (anti-HSG), 734 (anti-In-DTPA), L19 (anti-ED-B fibronectin), R1 (anti-IGF-1R), PAM4 (anti-MUC1), RS7 (anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab. 
     
     
         4 . The fusion protein of  claim 1 , wherein the antibody or antigen-binding fragment thereof binds to an antigen 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, EGFR, EGP-1, EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, hypoxia inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (ILGF-1), ILGF-1 receptor, KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, 5100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis factor-α, tumor-necrosis factor-β, VEGF, ED-B fibronectin, and 17-1A-antigen. 
     
     
         5 . The fusion protein of  claim 1 , wherein the antibody or antigen-binding fragment thereof is chimeric, humanized or human. 
     
     
         6 . The fusion protein of  claim 1 , wherein the antibody fragment is selected from the group consisting of Fab, Fab′, Fv, sFV and scFV antibody fragments 
     
     
         7 . The fusion protein of  claim 1 , wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α, tumor necrosis factor-β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); NGF-β; platelet-growth factor; TGF-α, TGF-β, erythropoietin (EPO); macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, FLT-3, angiostatin, thrombospondin, endostatin and lymphotoxin. 
     
     
         8 . The fusion protein of  claim 1 , wherein the cytokine is selected from the group consisting of G-CSF, interferon-β1A, interferon-α2b and erythropoietin. 
     
     
         9 . A fusion protein comprising: (i) an antibody, an antigen-binding antibody fragment or a cytokine conjugated to (ii) an anchoring domain (AD) moiety of an A-kinase anchoring protein (AKAP). 
     
     
         10 . The fusion protein of  claim 9 , wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (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), 734 (anti-In-DTPA), L19 (anti-ED-B fibronectin), R1 (anti-IGF-1R), PAM4 (anti-MUC1), RS7 (anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab. 
     
     
         11 . The fusion protein of  claim 9 , wherein the antibody or antigen-binding fragment thereof binds to an antigen 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, EGFR, EGP-1, EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, hypoxia inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (ILGF-1), ILGF-1 receptor, KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis factor-α, tumor-necrosis factor-β, VEGF, ED-B fibronectin, and 17-1A-antigen. 
     
     
         12 . The fusion protein of  claim 9 , wherein the antibody or antigen-binding fragment thereof is chimeric, humanized or human. 
     
     
         13 . The fusion protein of  claim 9 , wherein the antibody fragment is selected from the group consisting of Fab, Fab′, Fv, sFV and scFV antibody fragments 
     
     
         14 . The fusion protein of  claim 9 , wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α, tumor necrosis factor-β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); NGF-β; platelet-growth factor; TGF-α, TGF-β, erythropoietin (EPO); macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, FLT-3, angiostatin, thrombospondin, endostatin and lymphotoxin. 
     
     
         15 . The fusion protein of  claim 9 , wherein the cytokine is selected from the group consisting of G-CSF, interferon-β1A, interferon-α2b and erythropoietin. 
     
     
         16 . A dock-and-lock (DNL) complex comprising:
 a) a first fusion protein comprising (i) a first antibody, antigen-binding antibody fragment or cytokine conjugated to (ii) a dimerization and docking domain (DDD) moiety of human protein kinase A regulatory subunit RIα, RIβ, RIIα and RIIβ; and   b) a second fusion protein comprising (i) a second antibody or antigen-binding antibody fragment conjugated to (ii) an anchoring domain (AD) moiety of an A-kinase anchoring protein (AKAP);   wherein two copies of the DDD moiety a dimer and bind to the AD moiety to form the DNL complex.   
     
     
         17 . The DNL complex of  claim 16 , wherein the amino acid sequence of the DDD moiety is selected from the group consisting of residues 1 to 44 of human PKA RIIα, residues 1 to 44 of human PKA RIIβ, residues 12 to 61 of human PKA RIα and residues 13 to 66 of human PKA RIβ. 
     
     
         18 . The DNL complex of  claim 16 , wherein each antibody or antigen-binding fragment thereof is selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (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), 734 (anti-In-DTPA), L19 (anti-ED-B fibronectin), R1 (anti-IGF-1R), PAM4 (anti-MUC1), RS7 (anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab. 
     
     
         19 . The DNL complex of  claim 16 , wherein each antibody or antigen-binding fragment thereof binds to an antigen 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, EGFR, EGP-1, EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, hypoxia inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (ILGF-1), ILGF-1 receptor, KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis factor-α, tumor-necrosis factor-β, VEGF, ED-B fibronectin, and 17-1A-antigen. 
     
     
         20 . The DNL complex of  claim 16 , wherein each antibody or antigen-binding fragment thereof is chimeric, humanized or human. 
     
     
         21 . The DNL complex of  claim 16 , wherein each antibody fragment is selected from the group consisting of Fab, Fab′, Fv, sFV and scFV antibody fragments 
     
     
         21 . The DNL complex of  claim 16 , wherein each antibody fragment is selected from the group consisting of Fab, Fab′, Fv, sFV and scFV antibody fragments 
     
     
         22 . The DNL complex of  claim 16 , wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α, tumor necrosis factor-β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); NGF-β; platelet-growth factor; TGF-α, TGF-β, erythropoietin (EPO); macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, FLT-3, angiostatin, thrombospondin, endostatin and lymphotoxin. 
     
     
         23 . The DNL complex of  claim 16 , wherein the cytokine is selected from the group consisting of G-CSF, interferon-β1A, interferon-α2b and erythropoietin.