Methods for generating stably linked complexes composed of homodimers, homotetramers or dimers of dimers and uses
The present invention concerns methods and compositions for stably tethered structures of defined compositions, which may have multiple functionalities and/or binding specificities. Particular embodiments concern homodimers comprising monomers that contain a dimerization and docking domain attached to a precursor. The precursors may be virtually any molecule or structure, such as antibodies, antibody fragments, antibody analogs or mimetics, aptamers, binding peptides, fragments of binding proteins, known ligands for proteins or other molecules, enzymes, detectable labels or tags, therapeutic agents, toxins, pharmaceuticals, cytokines, interleukins, interferons, radioisotopes, proteins, peptides, peptide mimetics, polynucleotides, RNAi, oligosaccharides, natural or synthetic polymeric substances, nanoparticles, quantum dots, organic or inorganic compounds, etc. Other embodiments concern tetramers comprising a first and second homodimer, which may be identical or different. The disclosed methods and compositions provide a facile and general way to obtain homodimers, homotetramers and heterotetramers of virtually any functionality and/or binding specificity.
1. A tetramer comprising two homodimers, each homodimer comprising two copies of a fusion protein, each fusion protein comprising a human protein kinase A (PKA) regulatory subunit dimerization and docking domain (DDD) moiety, wherein the amino acid sequence of the DDD moiety is SEQ ID NO:1, the DDD moiety attached to an antigen-binding antibody fragment, wherein the two homodimers are covalently attached to each other by disulfide bonds between the DDD moieties.
2. The tetramer of claim 1 , wherein the two homodimers are identical.
3. The tetramer of claim 1 , wherein the two homodimers are different.
4. The tetramer of claim 1 , further comprising a linker peptide between the DDD moiety and the antigen-binding antibody fragment.
5. The tetramer of claim 1 , wherein the antibody fragment is selected from the group consisting of hMN-14 (anti-CEACAM5), hA20 (anti-CD20), hLL2 (anti-CD22), hLL1 (anti-CD74), hPAM4 (anti-MUC1), anti-CD14 and anti-CD111.
6. The tetramer of claim 1 , wherein the antigen-binding antibody fragment is selected from the group consisting of a Fab fragment, a Fab′ fragment, a single domain antibody (DAB) and a single chain Fv (scFv).
7. The tetramer of claim 1 , wherein the antibody fragment 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, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, 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 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, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, and 17-1A-antigen.
8. A tetramer comprising two homodimers, each homodimer comprising two copies of a fusion protein, each fusion protein comprising a human protein kinase A (PKA) regulatory subunit dimerization and docking domain (DDD) moiety, wherein the amino acid sequence of the DDD moiety is SEQ ID NO:2, the DDD moiety attached to an antigen-binding antibody fragment, wherein the two homodimers are covalently attached to each other by disulfide bonds between the DDD moieties.
9. The tetramer of claim 8 , wherein the antigen-binding antibody fragment is selected from the group consisting of a Fab fragment, a Fab′ fragment, a single domain antibody (DAB) and a single chain Fv (scFv).
10. The tetramer of claim 8 , wherein the antibody fragment 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, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, 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, KS 1-4, 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, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, and 17-1A-antigen.
11. The tetramer of claim 8 , wherein the antibody fragment is selected from the group consisting of hMN-14 (anti-CEACAM5), hA20 (anti-CD20), hLL2 (anti-CD22), hLL1 (anti-CD74), hPAM4 (anti-MUC1), anti-CD14 and anti-CD111.
12. The tetramer of claim 8 , wherein the two homodimers are identical.
13. The tetramer of claim 8 , wherein the two homodimers are different.