JP5231231B2 - Methods and compositions and uses for generating bioactive assemblies of increased complexity - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is based on the international application PCT / US / 2006/010762 filed on March 24, 2006 and the international application PCT / US / 2006/012084 filed on March 29, 2006. U.S. Patent Application No. 11 / 389,358, filed March 24, 2006, and U.S. Patent Application No. 11 / 391,584, filed Mar. 28, 2006 US Provisional Patent Application No. 60 / 728,292, filed on October 19, 2005, and US Provisional Patent Application No. 60, filed on December 16, 2005, in accordance with section 119 (e) of the United States Patent Law. All of the cited references, with allegations of priority under US Provisional Patent Application No. 60 / 782,332, filed March 14, 2006 The contents of are incorporated herein by reference.

FIELD OF THE INVENTION Various embodiments of the present invention relate to methods and compositions for making and using multivalent, multiple, and / or multifunctional assemblies. Such assemblies include, but are not limited to, cancers, autoimmune diseases, cardiovascular diseases, metabolic diseases, degenerative diseases including neurological diseases such as Alzheimer's disease, and transplanted organ rejection, infections, diseases, And uses in a wide range of applications in the field of treatment, detection and / or diagnosis of health related conditions.

BACKGROUND OF THE INVENTION Artificial agents that incorporate multiple sets of target and effector sites together are highly desirable because they provide stronger binding and greater efficacy. Recombinant techniques are commonly used to create fusion proteins containing both target and effector domains, but multimeric structures containing the same or different monomeric components can only be made by careful application of conjugation chemistry. Can be obtained.

  For drugs obtained by recombinant technology, high production costs, low expression yield, instability in serum, instability in solution leading to aggregate formation or subunit dissociation, multiple product forms Problems such as uncertainties in batch composition due to the presence of odors, contamination of by-products, reduced functional activity or affinity / binding properties due to changes in steric factors or conformation can occur. For drugs obtained by various chemical cross-linking methods, high manufacturing costs and heterogeneity of the purified product are two major constraints.

  Therefore, multispecific structures with multispecificity or multifunctionality that can be produced in high yield with constant composition, constant purity, without changing affinity, and without excessive purification. There is still a need for common manufacturing methods. Furthermore, the structure must be sufficiently stable in serum for in vivo application. There is also a need for stable multivalent structures with multispecificity or multifunctionality that are easy to construct and / or easily obtained in relatively high purity.

Summary The present invention discloses a base technology for generating highly complexing bioactive assemblies suitable for in vivo and in vitro applications. The assemblies are US Provisional Patent Application No. 60 / 728,292, filed Oct. 19, 2005, US Provisional Patent Application No. 60 / 751,196, filed Dec. 16, 2005, March 14, 2006. U.S. Provisional Patent Application No. 20 / 782,332, and U.S. Patent Application No. 11 / 389,358, filed March 28, 2006, the entire contents of each of which are hereby incorporated by reference. ) And recently reported (Rossi et al., Proc Natl Acad Sci USA, 2006, 103: 6841-6846) using a strategy based on the Dock and Lock (DNL) method or at least two different proteins or It is formed by regiospecific binding of non-proteins.

  Use of the bioactive assembly includes detection, diagnosis, and / or treatment of a disease or other related medical condition. The diseases include cancer, hyperplasia, diabetic retinopathy, macular degeneration, inflammatory bowel disease, Crohn's disease, ulcerative colitis, systemic rheumatism, diabetes, sarcoidosis, asthma, edema, pulmonary hypertension, psoriasis, corneal transplant rejection Reaction, neovascular glaucoma, hereditary hemorrhagic telangiectasia, myocardial neovascularization, plaque angiogenesis, restenosis, intimal neovascularization after vascular injury, telangiectasia, hemophilic arthropathy, angiofibroma, Examples include, but are not limited to, fibrosis associated with chronic inflammation, pulmonary fibrosis, amyloidosis, Alzheimer's disease, organ transplant rejection, deep vein thrombosis, or wound granulation.

  In certain embodiments, the disclosed methods and compositions comprise acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, sydnam chorea, myasthenia gravis, systemic lupus erythematosus, lupus Nephritis, rheumatic fever, multiglandular syndrome, bullous pemphigoid, juvenile diabetes, Henoch-Schenlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis Disease, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, obstructive thromboangiitis, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto Thyroiditis, thyroid poisoning, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomas, membranous nephropathy, muscle atrophy Can be used to treat autoimmune diseases such as multiple lateral sclerosis, spinal cord fistula, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, and fibroalveolitis .

  In certain embodiments, the bioactive assembly can be used to treat cancer. It is anticipated that any type of tumor and any type of tumor antigen can be targeted. Examples of types of tumors that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrium Cancer, esophageal cancer, stomach cancer, head and neck cancer, Hodgkinson lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkinson lymphoma, multiple myeloma, kidney cancer, ovarian cancer, pancreatic cancer, melanoma, liver cancer, prostate cancer, nerve Glioma and other brain tumors, as well as spinal cord tumors and bladder cancer.

  Tumor-associated antigens that can be targeted include carbonic anhydrase IX, A3, A33 antibody specific antigen, BrE3 antigen, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA95, NCA90, HCG and its subunits, CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba733 , HER2 / neu, hypoxia inducible factor (HIF), KC4-antigen, KS-1 antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, MUC16, PAM -4-antigen, PSA, SMA, RS5, S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growth factor-1 (IGF-1), Tn antigen, Thomsen-Friedenreich antigen, tumor necrosis antigen, VEGF, placental growth factor (PlGF), 17-1A antigen, angiogenesis markers (such as ED-B fibronectin), oncogene markers (such as bcl-2), oncogene products, and other tumor-associated antigens. Recent reports on tumor-associated antigens include Mizukami et al. (2005, Nature Med. 11: 992-97), Hatfield et al. (2005, Curr. Cancer Drug Targets 5: 229-48), Vallbohmer et al. (2005, J. Clin. Oncol. 23: 3536-44), and reports by Ren et al. (2005, Ann. Surg. 242: 55-63), each of which is incorporated herein by reference.

  In other embodiments, the bioactive assembly can be used to treat infections by pathogens such as bacteria, viruses, fungi, or unicellular parasites. Examples of fungi that can be treated include Microsporum, Trichophyton, Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans ), Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis or Candida albican. Examples of viruses include human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus, human papilloma virus, hepatitis B virus, hepatitis C virus, Sendai virus, feline leukemia virus, reovirus, Poliovirus, human serum parvo-like virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, varicella-zoster virus, dengue virus, rubella virus, measles virus, adenovirus, human T cell leukemia virus, Epstein-Barr A virus, murine leukemia virus, epidemic parotitis virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, or bluetongue virus. Examples of bacteria include Bacillus anthracis, group B streptococci (Streptococcus agalactiae), Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, medulla Neisseria meningitides, Pneumococcus spp., Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Hansen Examples include Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, or Mycoplasma. Examples of parasites include the Rumble ciliate (Giardia lamblia), Giardia spp., Pneumocystis carinii, Toxoplasma gondii, Cryptospordium spp., Acanthamoeba Genus (Acanthamoeba spp.), Neegleria spp., Leishmania spp., Colonic barantidium coli, Evans trypanosoma evansi, Trypanosoma spp., Binuclear amoeba (Dientamoeba fragilis), Trichomonas vaginalis, Trichomonas spp., Entamoeba spp., Binuclear amoeba (Dientamoeba spp.), Babesia spp., P. falciparum Protozoa (Plasmodium falciparum), Isospora spp., Toxoplasma (To xoplasma spp.), Enterocytozoon spp., Pneumocystis spp., and Balantidium spp.

  In various embodiments, but not limited to, bacterial toxins, plant toxins, ricin, abrin, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin (Pseudomonas exotoxin) exotoxin), Pseudomonas endotoxin, Lampyrinase (Rap), Rap (N69Q), PE38, dgA, DT390, PLC, tPA, cytokine, growth factor, soluble receptor component, surfactant protein D, IL-4 , SIL-4R, sIL-13R, VEGF121, TPO, EPO, thrombolytic agent, enzyme, fluorescent protein, sTNFα-R, avimer, scFv, dsFv, or peptide therapeutic or diagnostic agent such as nanobody The physiological activity Nburi to be bound, or may be incorporated.

  In certain embodiments, the anti-angiogenic agent may form part of or bind to the bioactive assembly. Examples of anti-angiogenic agents used include angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibody or peptide, anti-placental growth factor antibody or peptide, anti-Flk-1 antibody, anti-Flt-1 antibody or peptide , Laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxyestradiol, protein related to proliferin , Carboxamide triazole, CM101, marimastat, pentosan polysulfate, angiopoietin 2, interferon α, herbimycin A, PNU145156E, 16K prolactin fragment, linomay , Thalidomide, pentoxyferrin, genistein, TNP-470, endostatin, baclitaxel, accutin, angiostatin, cidofovir, vinsuristin, bleomycin, AGM-1470, platelet factor 4 or minocycline.

  In yet another embodiment, aplidine, azaribine, anastrozole, azacitidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamicin, camptothecin, 10-hydroxycamptothecin, carmustine, cereblex, chlorambucil, cisplatin, irinotecan ( CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomy single clonide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, 2-pyrrolinodoxorubicin (2P-) DOX), cyanomorpholino doxorubicin, doxorubi single clonide, epirubi single clonide, ethinyl es Radiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3 ′, 5′-O-dioleoyl FudR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine , Hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine, mechloretamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, Misramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-34 , Semestine, streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade, vinblastine, vinorelbine, vinristine, lysine, abrin, ribonuclease, L One or more therapeutic agents such as staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, Pseudomonas aeruginosa endotoxin, antisense oligonucleotides, or combinations thereof are physiologically active It may be coupled to or incorporated into the assembly.

  In various embodiments, for diagnostic agents, therapeutic agents, chemotherapeutic agents, radioisotopes, imaging agents, anti-angiogenic agents, cytokines, chemokines, growth factors, pharmaceuticals, prodrugs, enzymes, binding molecules, cell surface receptors Ligand, chelator, immunomodulator, oligonucleotide, interfering RNA, aptamer, hormone, photolabel, dye, peptide, toxin, contrast agent, paramagnetic label, ultrasound label, apoptosis promoter, liposome, nanoparticle, or One or more types of effectors such as combinations thereof may be bound to the bioactive assembly.

  Various embodiments may relate to bioactive assemblies and uses thereof that are useful for inducing apoptosis of abnormal cells. More details are described in US Patent Application Publication No. 20050079184, the entire contents of which are hereby incorporated by reference. The structure is CD2, CD3, CD8, CD10, CD21, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CEA, CSAp, CA-125, TAG-72, EFGR, HER2, HER3, HER4, IGF-1R, c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, MUC16, TNFR1, TNFR2, NGFR, Has affinity for an antigen selected from the group consisting of Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, ED-B fibronectin, tenascin, PSMA, PSA, carbonic anhydrase DC, and IL-6 That, fragments, etc. of the antibody or antibody may have a first and / or second coupling group. In more specific embodiments, bioactive assemblies useful for inducing apoptosis may include monoclonal antibodies, Fab fragments, chimeric antibodies, humanized antibodies, or human antibodies or fragments. In a preferred embodiment, the bioactive assembly is anti-CD74 antibody x anti-CD20 antibody, anti-CD74 antibody x anti-CD22 antibody, anti-CD22 antibody x anti-CD20 antibody, anti-CD20 antibody x anti-HLA-DR antibody, anti-CD19 antibody x anti-CD20. It may have a combination of antibodies, anti-CD19 antibody x anti-CD22 antibody, anti-CD20 antibody x anti-CD80 antibody, anti-CD2 antibody x anti-CD25 antibody, anti-CD8 antibody x anti-CD25 antibody, and anti-CD2 antibody x anti-CD147 antibody . In a more preferred embodiment, the chimeric, humanized, or human antibody or antibody fragment is derived from the variable domains of LL2 (anti-CD22 antibody), LL1 (anti-CD74 antibody), and A20 (anti-CD20 antibody). It may be.

  In certain embodiments, proteins or peptides with therapeutic effects well known in the art can be conjugated to AD or DDD sequences and used as effectors in the methods and compositions of the invention. Many proteins or peptides having such therapeutic effects are known, and are described in, for example, US Patent Application Publication No. 20060084794 "Albumin fusion proteins" filed on November 2, 2005, and all the contents thereof. Is incorporated herein by reference. Table 1 of US 20060084794 lists various known examples of useful proteins or peptides having therapeutic effects, with example names, patent reference numbers, and preferred indications, in particular The entire contents of which are hereby incorporated by reference. Useful other proteins or peptides having therapeutic effects are described, for example, in US Pat. No. 6,309,633, the entire contents of which are hereby incorporated by reference. Adrenocorticotropic hormone, shrimpide, angiotensin, angiotensin II, asparaginase, atrial natriuretic peptide, atrial natriuretic peptide, bacitracin, β-endorphin, blood coagulation factor VII, VIII, and IX, blood thymus factor, bone morphogenetic factor , Bone morphogenetic factor, bradykinin, cerulein, polypeptide related to calcitonin gene, calcitonin, CCK-8, cell growth factor, EGF, TGF-α, TGF-β, acidic FGF, basic FGF, chemokine, cholecystokinin , Cholecystokinin-8, cholecystokinin-pancreosamimi, colistin, colony stimulating factor, GMCSF, MCSF, corticotropin releasing factor, cytokine, desmopressin, dipeptide, dismutase, dynorphin , Eledoisin, endorphin, endothelin, endothelin-antagonist peptide, endothelin, enkephalin, epidermal growth factor, erythropoietin, vesicle stimulating hormone, galanin, gastric inhibitory polypeptide, gastric juice releasing polypeptide, gastrin, G-CSF, glucagon, glutathione peroxidase, Glutathione peroxidase, gonadotropin, gramicidin, gramicidins, growth factor, growth hormone releasing factor, growth hormone, h-ANP hormone releasing hormone, human chorionic gonadotropin, human chorionic gonadotropin β chain, human placental lactogen, insulin, insulin-like growth Factor, IGF-I, IGF-II, interferon, interleukin, intestinal polypeptide, kallikrein, kyotorphin, Ruri Phosphorus, luteinizing hormone, luteinizing hormone releasing hormone, lysozyme chloride, melanocyte stimulating hormone, melanocyte stimulating hormone, melittin, motilin, muramyl, muramyl dipeptide, nerve growth factor, neurotrophic factor, NT-3, NT-4 , CNTF, GDNF, BDNF, neuropeptide Y, neurotensin, oxytocin, pancreatinstatin, pancreatin polypeptide, pancreodimine, thyroid hormone, pentagastrin, polypeptide YY, pituitary adenyl cyclase activation polypeptide, platelet-derived Growth factor, polymyxin B, prolactin, protein synthesis stimulating polypeptide, PTH related protein, relaxin, renin, secretin, serum thymus factor, somatomedin, somatostatin, substance P, superoxide, -Peroxide dismutase, tuftsin, tetragastrin, thrombopoietin, thymic fluid factor, thymopoietin, thymosin, thymostimulin, thyroid hormone releasing hormone, thyroid stimulating hormone, thyrotropin releasing hormone (TRH), trypsin, tuftsin, tumor growth factor, tumor necrosis factor, Examples include, but are not limited to, thyroxine, urogastrin, urokinase, vasoactive intestinal polypeptide, vasopressin, and functional equivalents.

DESCRIPTION OF SPECIFIC EMBODIMENTS All references or parts of references cited in this application, including but not limited to patent publications, patent applications, articles, books, and articles, are expressly incorporated in their entirety. Which is incorporated herein by reference.

Definitions As used herein, “a” or “an” may mean one or more things.

  As used herein, “and” and “or” can both be used in both conjunctive or disjunctive meanings. That is, any term is understood to be equivalent to “and / or” unless stated otherwise.

  As used herein, an antibody refers to a full-length immunoglobulin molecule (such as an IgG antibody) (natural or formed by a recombination process of ordinary immunoglobulin gene fragments), or an antibody fragment or the like. It refers to an immunologically active (specific binding) site or analog of a globulin molecule.

The antibody fragment is a part of an antibody such as F (ab) ₂ , F (ab ′) 2, Fab, Fv, sFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the normal antibody. The term “antibody fragment” includes any synthetic or genetically engineered protein that behaves as an antibody by binding to a specific antigen to form an assembly. For example, an antibody fragment is a recombinant single-chain polypeptide in which a variable domain such as an “Fv” fragment consisting of a heavy chain and a light chain variable domain, a light chain and a heavy chain are connected by a peptide linker (“scFv protein”). It includes an isolated fragment consisting of a molecule and a minimal recognition unit (CDR) consisting of amino acid residues that mimic the hypervariable domain.

  An effector is an atom, molecule, or compound that produces a selected result. The effector can include a therapeutic agent and / or a diagnostic agent.

  A therapeutic agent is an atom, molecule, or compound that is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, pharmaceuticals, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, short interfering RNA (siRNA), aptamers, chelating agents, boron compounds, photoactivity Agents, dyes, and radioisotopes. Other examples of therapeutic agents and methods of use are described in US Patent Application Publication Nos. 20050002945, 20040018557, 20030148409, and 2005014207, each of which is incorporated herein by reference.

  A diagnostic agent is an atom, molecule, or compound that is useful in diagnosing a disease by in vitro or in vivo testing. Examples of useful diagnostic agents include radioisotopes, dyes (such as those based on biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and sensitizers for magnetic resonance imaging (MRI) (paramagnetic) Ions) and the like, but is not limited thereto.

  An immunoconjugate is a conjugate of a binding molecule (such as an antibody component) and an atom, molecule, or higher-order structure (such as a carrier, therapeutic agent, or diagnostic agent).

  Naked antibodies are antibodies that are not bound to other drugs.

  A carrier is an atom, molecule, or higher order structure that can associate with therapeutic or diagnostic agents to increase the delivery of these agents to cells. Carriers include proteins, peptides, lipids (such as amphiphilic lipids that can form higher order structures), polysaccharides (such as dextrans), or other higher order structures such as micelles, liposomes, or nanoparticles. It is done.

  As used herein, an antibody fusion protein refers to two or more identical or different scFv or antibody fragment recombinantly produced antigen-binding molecules to which the same or different species are bound. The valence of the fusion protein indicates the number of bonds or sites (monovalent, divalent, trivalent, or multivalent) contained in one antigen or epitope. The multivalent nature of an antibody fusion protein means that the antibody fusion protein can utilize multiple interactions when binding to an antigen, thus increasing the strength of binding to the antigen. Specificity indicates the number of antigens or epitopes (monospecific, bispecific, trispecific, multispecific) that can bind to the antibody fusion protein. Using these definitions, natural antibodies (such as IgG) are bivalent because they have two bonds, but are monospecific because they bind to one epitope. A monospecific multivalent fusion protein has more than one bond to an epitope, but binds to only one of the epitopes, eg, has two binding sites that bind the same antigen It is a diabody. A fusion protein may comprise a single antibody component, a combination of different antibody components having multivalency or multispecificity, or multiple identical antigen components. The fusion protein may further comprise an antibody or antibody fragment and a therapeutic agent. Examples of such therapeutic agents suitable for fusion proteins are immunomodulators (“immunomodulator fusion proteins”), toxins (“antibody-toxin fusion proteins”), and the like. One preferred toxin comprises ribonuclease (RNase), preferably recombinant RNase.

  If the amount administered has a physiological effect, the antibody or immunoconjugate formulation, or composition described herein is said to have been administered "a therapeutically effective amount". An agent is physiologically effective if its presence results in a detectable change in the physiology of the administered mammal. In particular, antibody formulations are physiologically effective if their presence causes an anti-tumor response or alleviates signs or symptoms of an autoimmune disease state. A physiologically important effect may be to elicit a humoral and / or cellular immune response that results in target cell degeneration, growth inhibition, or death in the administered mammal.

DNL-Based Bioactive Assemblies Certain embodiments of the present invention may relate to bioactive assemblies formed using a position-specific binding strategy based on the Dock-and-Lock (DNL) method. The DNL method utilizes α-helix peptides that have been found to have the property of binding specifically to each other. α-helix peptides are dimerization and docking domains (DDD) in the regulatory (R) subunit of cAMP-dependent protein kinase (PKA), and the anchor domains of various A-kinase anchor proteins (AKAP) (AKAP). AD). When individual peptides are recombinantly fused or chemically conjugated to a substance of interest, these helices “dock” the two modified substances to form a metastable structure, They are “locked” by disulfide bonds formed from cysteine residues introduced into these helices, providing an excellent linker module for forming stable complexes. In PKA, two types of R subunits (RI and RII) have been identified and have α and β isoforms, respectively. Since the R subunit is isolated only as a stable dimer, and AKAP binds only to the dimer of the R subunit, the unique feature of the DNL method is a substance having a peptide derived from DDD Always form a homodimer, and there are two sets of the substance in the final complex.

  Two sets of interacting DDD and AD peptides are of particular interest as linker modules. The first set includes DDD2 (FIG. 1A, SEQ ID NO: 1) derived from the 44 amino terminal residues of human RIIa and AKAP-ZS, a synthetic protein optimized for selective binding to RIIa. (Alto et al., Proc Natl Acad Sci USA, 2003, 100: 4445-4450) and AD2 (FIG. 1B, SEQ ID NO: 2). The second set consists of DDD3 (FIG. 1C, SEQ ID NO: 3) or DDD3C derived from the peptide fragment (residues 12-61) of human RIa (Leon et al., J Biol Chem, 1997, 272: 28431-28437). (FIG. 1D, SEQ ID NO: 4) and derived from PV-38 (Burns-Hamuro et al., Proc Natl Acad Sci USA, 2003, 100: 4072-4077), a mutant peptide of D-AKAP2 that specifically binds to RIa. AD3 (FIG. 1E, SEQ ID NO: 5).

In one embodiment, two distinct AD peptides, one selectively binds to RIIα DDD (eg, AD2 is DDD2) and the other selectively binds to RIα DDD (eg, AD3 is DDD3C). type a adapter module comprising (Ma) and thereafter to produce a biological substance called (denoted by X ₂₎ is one using it, homodimer with each monomeric subunit that binds to the DDD of RIIα A complex with two other biological substances, referred to as peripheral modules in the future, consisting of different homodimers (denoted Y ₂ ) with each monomer subunit binding to the DDD of RIα. As a result, as shown in FIG. 2, an assembly X ₂ (Ma) Y ₂ including five types of components is formed.

In another embodiment, a type-b adapter module comprising two separate DDD peptides, one (eg DDD2) selectively reacting with AD2 and the other (eg DDD3C) selectively reacting with AD3 ( Mb) and a future biological substance called “Mb” ₂ is produced as a homodimer, and using it, one of them consists of a monomeric subunit (designated X) bound to AD2. The other forms a complex with two peripheral modules consisting of another monomeric subunit (designated Y) bound to AD3, resulting in four components as shown in FIG. As a result, an assembly X (Mb) ₂ Y is formed.

In yet another embodiment, a biological material, hereinafter referred to as a type c adapter module (Mc), comprising both AD2 and DDD3, is produced as a homodimer, hereinafter referred to as (Mc) ₂ and used. each, to form a complex of two identical peripheral modules consisting homodimer with each monomeric subunit bound to DDD2 (X ₂ hereinafter), as a result, as shown in FIG. 4 Will form an assembly X ₂ (Mc) ₂ X ₂ containing six components.

In a further embodiment, a biological material, hereinafter referred to as a type d adapter module (Md) comprising both AD2 and DDD3C (instead of DDD3 as in type c), a homodimer, hereinafter referred to as (Md) ₂ manufactured as, two therewith, at the same homodimer (X ₂ hereinafter) having each monomeric subunit bound to DDD2, the third one, the monomers linked to AD3 Form a complex with three peripheral modules consisting of subunits (denoted Y), resulting in an assembly X ₂ (Md) ₂ YX ₂ containing seven components, as shown in FIG. Will do.

  In certain embodiments, the bioactive assembly produced according to the present invention may be further coupled to an effector and a carrier to add functionality that can be obtained by the modification. Furthermore, bioactive assemblies can be DNA or RNA, or synthetic oligonucleotides (ODN) containing immunostimulatory CpG motifs (Klinman, Nat Rev Immunol, 2004, 4: 1-10; Krieg, Nat Rev Drug Discov, 2006, 5: 471-484) and may be configured to include components capable of binding.

  The methods and compositions of the present invention can produce a large number of bioactive assemblies with a wide range of applications depending on the type of adapter module selected and the types of peripheral modules attached to the adapter module. Bioactive substances that are of particular interest as adapter modules include human IgG1 Fc domain, human serum albumin (HSA), various heat shock proteins (HSP), bioluminescent proteins, human transferrin (hTf), and human protamine. Bioactive substances that can be induced to act as peripheral modules include cytokines, chemokines, growth factors, soluble receptors, antibody fragments, fluorescent proteins, l-peptides, d-peptides, peptides containing unnatural amino acids, Peptoid, mimetic peptide, DNA sequence, synthetic CpGODN, short interfering RNA, human protamine 1, DNA-binding peptide derived from protamine, protein conversion domain, nuclear localization signal, peptide that increases percutaneous absorption or membrane penetration DNA and RNA aptamers, peptide aptamers, cholera toxin subunit B monomers, enzymes, polyethylene glycol, nanoparticles, drug-containing polymers, chelates, quantum dots, and nanobodies, shrimp bodies, ankyrin repeat proteins, transbodies -Anticalins, microbodies, adnectin domain antibodies, affibodies, maxibodies, tetranectins, affilin molecules, iMabs, and Monobodies (Hey et al., Trends Biotechnol, 2005, 23: 514-522; Binz et al., Nat Biotechnol, 2005, 23: 1257-1268) and other binding proteins based on various structures. Specific compositions of selected assemblies based on adapter modules of type a, b, c, and d are shown in Tables 1, 2, 3, and 4, respectively.

Adapter module based on HSP A subunit vaccine consisting of well-characterized molecules is very promising because of its excellent safety profile and ease of manufacture, but it is poorly immunogenic and poorly stable Can be improved by the development of improved delivery vehicles and auxiliary agents that are highly efficient but non-toxic. The compositions and methods of the present invention comprise (1) a specific antigenic molecule, (2) a built-in adjuvant to increase the immune response, and (3) antigen-specific T cell immunity. It can be applied to the production of subunit vaccines that can be induced.

  One approach is to combine peripheral modules derived from target antigens and immunostimulants, which are proteins or peptides, to produce HSP-based type a adapter modules, resulting in direct immunization or ex vivo trees. Resulting in vaccines based on proteins or peptides for dendritic cell priming and realizing antigen presentation by both HC-I and MHC-II (Srivastava, Nat Rev Immunol, 2002, 2: 185-194 ). Alternatively, an HSP-based aptamer module can be combined with a DNA binding protein, such as human protamine (Song et al., Nat Biotechnol, 2005, 23: 709-717), or a clustered arginine residue, RRRRRRGGRRRRRRR (SEQ ID NO: 10) (Brewer et al., J Biol Chem, 2003, 278: 42403-42406) and the like, and binding to peripheral modules derived from target molecules such as antibody fragments and the like, target genes, or target genes and immune promoters A multifunctional assembly useful as a target-specific DNA vaccine is obtained by conjugation with a plasmid encoding both of them. In addition, HSP has the ability to non-covalently associate with various antigenic peptides (US Pat. No. 5,935,576 and US Pat. No. 5,750,119), thus depending on the vaccine It has the potential to broaden the spectrum for broader protection. Example 1 describes the generation and use of a type a adapter module based on HSP.

Adapter Module Based on Human Protamine Bioactive assemblies based on human protamine are particularly useful for gene delivery with therapeutic effects on DNA vaccines, siRNAs and specific cells. A fusion protein consisting of an anti-gp120 Fab antibody and human protamine 1 (hP1) (F105-P), which encodes Pseudomonas aeruginosa exotoxin A (Chen et al., Gene Ther, 1995, 2: 116-123), or siRNA Have been shown to be effective for delivery to HIV-infected cells or tumor cells expressing HIV envelope (Song et al., Nat Biotechnol, 2005, 23: 709-717). In order to bind peripheral modules derived from different biological materials such as proteins that specifically bind to the target, an adapter module of type b based on hP1 can be generated and the assembly thus obtained is complexed Can be used for target-specific delivery of siRNA, a plasmid that binds to hP1. Alternatively, a type c adapter module based on hP1 can be generated to bind peripheral modules derived from the same biological material, such as a protein that specifically binds to the target, and the four sets thus obtained Assemblies having proteins that specifically bind to the target can be used for target-specific delivery of plasmids, siRNA, that bind to hP1 by conjugation. In a further embodiment, a type d adapter module based on hP1 can be generated to combine peripheral modules derived from two different biological materials, and thus four sets of single materials obtained And an assembly comprising a set of other substances can be used for target-specific delivery of siRNA, a plasmid that binds to hP1 by conjugation. Examples 2 and 3 describe the generation and use of type b and type c adapter modules based on hP1, respectively.

Adapter Modules Based on the Fc Domain of Human Immunoglobulins Fusion proteins containing the Fc domain of human IgG have many advantages due to the inherent nature of the Fc domain. For example, Fc domain binding to the neonatal receptor (FcRn) expressed in lung and small intestinal epithelium facilitates transport of Fc fusion proteins across the mucosal barrier (Spiekermann et al., J Exp Med, 2002, 196: 303- 310), enabling delivery to the lung or oral cavity (Dumont et al., J Aerosol Med, 2005, 18: 294-303; Bitonti et al., Proc Natl Acad Sci USA, 2004, 101: 9763-9768; Low et al., Hum Reprod, 2005, 20: 1805-1813). Also, pH-dependent Fc binding to FcRn continuously expressed on the vascular endothelium increases the serum half-life of IgG antibodies or fusion proteins containing Fc. IgG or Fc domain variants with higher affinity for FcRn have been shown to significantly increase the half-life in serum of the modified construct (Hinton et al., J Immunol, 2006, 176: 346-356; Hinton et al., J Biol Chem, 2004, 279: 6213-6216). On the other hand, IgG or Fc domain variants with lower affinity for FcRn than the corresponding wild type showed shorter serum half-lives (Kenanova et al., Cancer Res, 2005, 65: 622- 631). The ability to modulate the pharmacokinetics of biological materials containing Fc domains is very attractive for drug design. A summary of the generation and use of type b, c, and d adapter modules based on Fc domains is shown in Examples 4, 5, and 6, respectively. A detailed description of the construction of expression vectors for DDD3-CH2-CH3-AD2 and DDD3C-CH2-CH3-AD2 is described in Example 7.

Conjugates of bioactive assemblies Other residues may be further conjugated to the bioactive assemblies described above. For example, drugs, toxins, radioactive compounds, enzymes, hormones, cytotoxic proteins, chelates, cytokines, and other active agents can be conjugated to the bioactive assembly. For example, it can be conjugated via a covalent bond to an amino acid residue containing an amine, carboxyl, thiol, or hydroxyl group in the side chain. For example, various linkers such as diisocyanate, diisothiocyanate, bis (hydroxysuccinimide), ester, carbodiimide, maleimide-hydroxysuccinimide ester, glutaraldehyde can be used for this purpose. Conjugation of the drug to the bioactive assembly preferably does not affect the activity of individual subunits contained in the unmodified structure. Coupling can be done separately for different peripheral modules and the resulting conjugates used for the preparation of the bioactive assembly. In addition, the cytotoxic agent may be first bound to the polymer carrier and then conjugated to the bioactive assembly. For this method, see Ryser et al., Proc. Natl. Acad. Sd. USA, 75: 3867-3870, 1978; U.S. Pat. No. 4,699,784, and U.S. Pat. No. 4,046,722. . Each incorporated herein by reference. As described below, one or more effectors may be attached to a carrier residue and then targeted to a bioactive assembly, for example by incorporation into a monoclonal antibody or fragment thereof that specifically binds to the carrier residue. Also good. Examples of the use of carrier residues to deliver effector molecules to bioactive assemblies localized to targeted cells, tissues, or pathogens are described in the Pre-Targeting section below.

The conjugates described herein can be prepared using various methods well known in the art. For example, the bioactive assembly can be radiolabeled with ¹³¹ I and the bioactive assembly can be conjugated to a lipid so that the resulting conjugate can form a liposome. Liposomes may contain one or more therapeutic agents (agents such as FUdR-dO) or diagnostic agents. Alternatively, in addition to the carrier, the bioactive assembly is conjugated to ¹³¹ I and a drug (eg, on the ε-amino group of lysine) (eg, on a tyrosine residue), and the carrier contains other therapeutic or diagnostic agents. May contain. The therapeutic and diagnostic agents may be covalently linked to one or more subunits of the bioactive assembly.

  The formation of liposomes and micelles is known. For example, Wrobel and Collins, Biochimica et Biophysica Acta (1995), 1235: 296-304; Lundberg et al., J. Pharm. Pharmacol. (1999), 51: 1099-1105; Lundberg et al., Int. J. Pharm. (2000 ), 205: 101-108; Lundberg, J. Pharm. Sci. (1994), 83: 72-75; Xu et al., Molec. Cancer Ther. (2002), 1: 337-346; Torchilin et al., Proc. Nat. 'l. Acad. Sci., USA (2003), 100: 6039-6044, US Pat. No. 5,565,215, US Pat. No. 6,379,698, and US Patent Application Publication No. 2003/0082154. Please refer.

  Nanoparticles or nanocapsules formed from polymers, silica, or metals and useful for drug delivery or imaging have also been described in the literature, for example, West et al., Applications of Nanotechnology to Biotechnology (2000), 11: 215- See 217 U.S. Pat. No. 5,620,708, U.S. Pat. No. 5,702,727, and U.S. Pat. No. 6,530,944. Conjugation of antibodies or binding molecules to liposomes to form target carriers for therapeutic or diagnostic agents and liposomes has been described in the literature, for example, Bendas, Biodrugs (2001), 15: 215-224; Xu et al. , MoI. Cancer Ther (2002), 1: 337-346; Torchilin et al., Proc. Nat'l. Acad. Sci. US A (2003), 100: 6039-6044; Bally et al., J. Liposome Res. (1998) ), 8: 299-335; Lundberg, Int. J. Pharm. (1994), 109: 73-81; Lundberg, J. Pharm. Pharmacol. (1997), 49: 16-21; Lundberg, Anti-cancer Drug See Design (1998), 13: 453-461. Also, U.S. Patent No. 6,306,393, U.S. Patent Application Publication No. 10 / 350,096, U.S. Patent Application Publication No. 09 / 590,284, and U.S. Provisional Patent Application No. 09 / 590,284 filed on June 9, 1999. See 60 / 138,284. Each is incorporated herein by reference.

  A wide range of diagnostic and therapeutic agents can be advantageously used to form conjugates of bioactive assemblies, or can bind to haptens that bind to recognition sites in bioactive assemblies. Diagnostic agents include radioisotopes, sensitizers for MRI or contrast agents for ultrasound imaging, and fluorescent compounds. Many suitable imaging agents are known along with methods for attaching them to proteins or peptides (see, eg, US Pat. Nos. 5,021,236 and 4,472,509, respectively). Is incorporated herein by reference). One conjugation method involves the use of a metal chelate complex using, for example, an organic chelating agent such as DTPA coupled to a protein or peptide (see US Pat. No. 4,472,509).

  In order to introduce a radioactive metal or paramagnetic ion into a bioactive assembly, it is first necessary to react with a carrier to which a plurality of chelating groups that bind to the radioactive metal or paramagnetic ion are bound. The carrier may be useful for such purposes as polylysine, polysaccharides or, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamine, crown ether, bisthiosemicarbazone, polyoxime, etc. It may be a polymeric derivative having a pendant group to which a known chelating group can be attached, or a derivatizable polymeric substance. The chelate-containing carrier binds to the bioactive assembly using standard chemistry that minimizes aggregation and loss of immune activity.

Other methods and reagents for preparing the conjugates are disclosed in US Pat. No. 4,824,659, the entire contents of which are hereby incorporated by reference. This particularly useful metal-chelate combination is 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs typically used with diagnostic isotopes with an energy range of 60-4000 keV. Some useful diagnostic nuclei include ¹²⁴ I, ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁴ Tc, ^{94 m} Tc, ^{99 m} Tc, Or ¹¹¹ In is mentioned. Similar chelate complexes with non-radioactive metals such as manganese, iron and gadolinium are useful for MRI when used with the bioactive assemblies and carriers described herein. Macrocyclic chelates such as NOTA, DOTA, and TETA are useful when used with many metals and radioactive metals, particularly gallium, yttrium, and copper radioactive nuclei, respectively. The metal-chelate complex can be very stabilized by adjusting the ring size with respect to the target metal. Other cyclic chelates such as macrocyclic polyethers for ²²³ Ra may be used.

  Therapeutic agents include, for example, vinca alkaloids, anthracyclines, epidophilotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotic agents, antiangiogenic agents and pro-apoptotic agents Chemotherapeutic agents such as agents, especially doxorubicin, methotrexate, taxol, CPT-11, SN-38, camptothecan, and other forms of these and other anticancer agents. Suitable chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), and their revised editions Has been. Other suitable chemotherapeutic agents such as experimental agents are well known to those skilled in the art and can be conjugated to the bioactive assemblies described herein using methods well known in the art.

Other types of therapeutic agents include α particles ( ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ²²³ Ra, ²²⁵ Ac, etc.), β particles ( ³² P, ³³ P, ⁴⁷ Sc, ⁶⁷ Cu, ⁶⁷ Ga, ⁸⁹ Sr, ⁹⁰ Y, ¹¹¹ Ag, ¹²⁵ I, ¹³¹ I, ¹⁴² Pr, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁶⁶ Dy, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, etc.) or Auger electrons ( ¹¹¹ In, ¹²⁵ I, ⁶⁷ Ga, ¹⁹¹ Os, ^{193 m} Pt, ^{195 m} Pt, ^{195 m} Hg, etc.). The bioactive assembly can be labeled with one or more radionuclei using the methods described for diagnostic agents.

  Proteins or peptides with therapeutic effects useful as effectors are described in US Pat. No. 6,309,633 (incorporated herein by reference), eg, adrenocorticotropic hormone (ACTH), adrenal gland Corticotropin derivatives (eg, shrimp), angiotensin, angiotensin II, asparaginase, atrial natriuretic peptide, atrial natriuretic peptide, bacitracin, β-endorphin, blood coagulation factor VII, VIII, and IX, blood thymus factor (FTS) , Blood clotting factor derivatives (see US Pat. No. 4,229,438), bombedin, bone morphogenetic factor (BMP), bone morphogenetic protein, bradykinin, cerulein, calcitonin gene related polypeptide (CGRP) , Cal Tonin, CCK-8, cell growth factor (EGF, TGF-α, TGF-β, PDGF, acidic FGF, basic FGF, etc.), cerulein, chemokine, cholecystokinin, cholecystokinin-8, cholecystokinin-pan Creosoimine (CCK-PZ), colistin, colony stimulating factor (CSF, GCSF, GMCSF, MCSF, etc.), corticotropin releasing factor (CRF), cytokine, desmopressin, dynorphin, dipeptide, dismutase, dynorphin, eledoisin, endorphin, Endothelin, endothelin-antagonist peptides (see European Patent Application Publication Nos. 436189, 457195, and 496452, and JP-A-3-94692 and JP-A-3-130299), Docerines, enkephalins, enkephalin derivatives (see US Pat. No. 4,277,394 and European Patent Application No. 31567), epidermal growth factor (EGF), erythropoietin (EPO), vesicle stimulating hormone (FSH) , Galanin, gastric inhibitory polypeptide, gastric juice releasing polypeptide, (GRP), gastrin, G-CSF, glucagon, glutathione peroxidase, glutathioperoxidase, gonadotropin (human chorionic gonadotropin and its α, β subunits, etc.), gramicidin, Gramicidins, growth factor (EGF), growth hormone releasing factor (GRF), growth hormone, hormone releasing hormone (LHRH), human atrial natriuretic peptide (h-ANP), human placental lactogen, insulin, in Phosphorus-like growth factor (IGF-I, IGF-II), interferon, interferon tears (α-, β-, and γ-interferon, etc.), interleukin (1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, and 12, etc.), intestinal polypeptide (VIP), kallikrein, kyotorphin, luriberin, luteinizing hormone (LH), luteinizing hormone-releasing hormone (LH-RH), lysozyme chloride, melanocyte stimulation Hormone (MSH), melanocyte stimulating hormone, melittin, motilin, muramyl, muramyl dipeptide, nerve growth factor (NGF), neurotrophic factor (NT-3, NT-4, CNTF, GDNF, BDNF, etc.), neuropeptide Y , Neurotensin, oxytocin, pancreatin, pancreatin polypeptide, pa Nucleodimine, thyroid hormone (PTH), pentagastrin, polypeptide YY, pituitary adenyl cyclase activation polypeptides (PACAPs), platelet-derived growth factor, polymyxin B, prolactin, protein synthesis stimulating polypeptide, relaxin protein, relaxin , Renin, secretin, serum thymic factor, somatomedin, somatostatin derivatives (sandstatin, US Pat. Nos. 4,087,390, 4,093,574, 4,100,117, and 4,253,998) ), Substance P, superoxide, superoxide dismutase, tuftsin, tetragastrin, thrombopoietin (TPO), thymic fluid factor (THF), thymopoietin, thymosin, thymostimulin, thyroid hormone release hormone , Thyrotropin releasing hormone (TSH), thyrotropin releasing hormone (TRH), trypsin, tuftsin, tumor growth factor (TGF-α), tumor necrosis factor (TNF), thioxidine, urogastrin, urokinase, vasoactive intestinal polypeptide, vasopressin And those functionally equivalent to the polypeptide.

  A suitable peptide containing a detectable label (such as a fluorescent molecule) or a cytotoxic agent (such as radioactive iodine) can be covalently coupled, non-covalently coupled, or otherwise bound to the bioactive assembly. For example, conjugates useful for therapy can be obtained by introducing photoactive agents or dyes into the bioactive assembly. Fluorescent compositions such as fluorescent dyes and other chromophores or dyes such as porphyrins that are sensitive to visible light are used to detect and treat lesions by irradiating the lesions with appropriate light. In therapy, the method is called light irradiation, phototherapy, or photodynamic therapy. See Jori et al. (Eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985), van den Bergh, Chem. Britain (1986), 22: 430. In addition, a monoclonal antibody was conjugated with a photoactive dye for phototherapy. Mew et al., J. Immunol. (1983), 130: 1473, ibid., Cancer Res. (1985), 45: 4380, Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83: 8744, ibid. Photochem. Photobiol. (1987), 46:83, Hasan et al., Prog. Clin. Biol. Res. (1989), 288: 471, Tatsuta et al., Lasers Surg. Med. (1989), 9: 422; Pelegrin et al., See Cancer (1991), 67: 2529. Application of endoscopes is also conceivable. Endoscopic detection and treatment methods are described in US Pat. No. 4,932,412, US Pat. No. 5,525,338, US Pat. No. 5,716,595, US Pat. No. 5,736,119. US Pat. No. 5,922,302, US Pat. No. 6,096,289, and US Pat. No. 6,387,350, the entire contents of which are hereby incorporated by reference. The

  In certain embodiments, the novel compositions and methods disclosed herein are useful for targeted gene transfer of RNAi for therapy. The introduction means may be a bioactive assembly fused to human protamine (a peptide of about 50 amino acid residues) and having an internalized antibody binding domain. Examples include human protamine 1 (hP1) and / or protamine 2 (hP2), both of which can form stable DNA or RNA complexes with RNAi etc. for in vivo use (Nat Biotechnol. 23: 709-717, 2005, Gene Therapy. 13: 194-195, 2006). The multivalent complex promotes binding and internalization to the target cell, and non-covalently bound RNAi dissociates inside the endosome and is released into the cytoplasm. In addition to RNAi delivery, these compositions are also useful for targeted gene transfer of therapeutically effective genes or DNA vaccines. Other applications include the application of this technology to the production of intracellularly expressed antibodies that are functionally RNAi protein analogs.

Administration of Peptides Various embodiments relating to the methods and / or compositions of the present invention relate to bioactive assemblies based on one or more peptides for administration to a subject. Administration includes oral, nasal, buccal, inhalation, rectal, vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intrathecal, or intravenous infusion, etc. Without being limited thereto, any method known in the art can be used.

  Unmodified peptides that are orally administered to a subject may be degraded in the digestive tract and, depending on the sequence and structure, may be poorly absorbed from the lining of the intestinal tract. However, methods for chemically modifying peptides to reduce the risk of degradation by endogenous enzymes and increasing absorption from the gastrointestinal tract are well known (eg, Blondele et al., 1995, Biophys. J. 69). : 604-11, Ecker and Crooke, 1995, Biotechnology 13: 351-69, Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY, VOL. I, Wollf (edited). John Wiley & Sons, Goodman and Shao, 1996 , Pure & Appl. Chem. 68: 1303-08). D-peptides containing amino acids, mimetic peptides consisting of organic molecules that mimic the peptide structure. Alternatively, methods for preparing a library of peptide analogs such as peptoids such as vinylpeptoids have also been described in the literature and can be used to construct peptide-based bioactive assemblies useful for oral administration to a subject.

In some embodiments, the standard peptide _{bond, CH 2 -NH, CH 2 -S} , CH 2 -CH 2, CH = CH, CO-CH 2, 1 or more other bonds such CHOH-CH ₂ May be substituted. Methods for preparing peptidomimetics are well known (eg, Hruby, 1982, Life Sci 31: 189-99, Holladay et al., 1983, Tetrahedron Lett. 24: 4401-04, Jennings-White et al., 1982, Tetrahedron Lett. 23: 2533, Almquiest et al., 1980, J. Med. Chem. 23: 1392-98, Hudson et al., 1979, Int. J. Pept. Res. 14: 177-185, Spatola et al., 1986, Life Sci 38 : 1243-49, U.S. Pat. Nos. 5,169,862, 5,539,085, 5,576,423, 5,051,448, 5,559,103. Each of which is incorporated herein by reference). Peptidomimetics may have improved in vitro stability and / or absorbability compared to analogs of those peptides.

  Alternatively, the peptide can be administered using N-terminal and / or C-terminal capping to inhibit exopeptidase activity. For example, the C-terminus can be capped with an amide peptide and the N-terminus can be capped by acetylation of the peptide. To block exopeptidase, the peptide may be cyclized, for example, by formation of cyclic amides, disulfides, ethers, sulfides, and the like.

  Peptide stabilization can be achieved in particular by substituting D-amino acids for natural L-amino acids at positions where endopeptidases are known to act. Sequences to which endopeptidase binds and degrades are well known in the art, and methods for producing and using peptides incorporating D-amino acids are described in the literature (eg, McBride et al., June 14, 2004). See U.S. Patent Application Publication No. 20050025709, which is hereby incorporated by reference, each of which is incorporated herein by reference). In certain embodiments, the peptides and / or proteins can be formulated and administered orally with inhibitors of proteolytic enzymes and / or peptidases.

  Another method for the oral administration of therapeutic peptides is disclosed by Mehta ("Oral delivery and recombinant production of peptide hormones" June 2004, BioPharm International). The peptide is administered as a solid dosage form with an enteric coating that regulates proteolytic enzyme activity in the intestine and increases the transport of the peptide through the intestinal wall. Using this approach, the relative bioavailability of the complete peptide is 1-10% of the dose. Insulin could be administered to dogs using enteric microcapsules with sodium cholate and protease inhibitors (Ziv et al., 1994, J. Bone Miner. Res. 18 (Suppl. 2) : 792-94). Oral administration of peptides was performed using acylcarnitine as a permeation enhancer and enteric coating (see Eudragit L30D-55, Rohm Pharma Polymers, Mehta, 2004). Excipients used for orally administered peptides usually include one or more intestinal protease / peptidase inhibitors, surfactants or other agents to promote peptide solubility or absorption. It may be encapsulated inside an enteric coated capsule or tablet (Mehta, 2004). After the capsule is dissolved in the intestine, an organic acid may be included in the capsule to acidify the intestine and inhibit intestinal protease activity. Another method for oral administration of peptides is by conjugation with an amphiphilic oligomer based on polyethylene glycol (PEG) to increase absorption and inhibit enzymatic degradation (Soltero and Ekwuribe, 2001, Pharm Technol. 6: 110).

  In yet another embodiment, it can be modified for oral and inhalation administration by conjugation with a specific protein such as the Fc domain of IgG1 (see Examples 3-7). For preparation and use of peptide-Fc domain conjugation, see, for example, Low et al. (2005, Hum. Reprod. 20: 1805-13) and Dumont et al. (2005, J. Aerosol. Med. 18: 294-303 Each of which is incorporated herein by reference. Low et al. (2005) disclose the complexation of FSH α and β subunits with single-chain or heterodimeric IgG1 Fc domains using recombinant expression in CHO cells. . Fc domain-conjugated peptides are absorbed into epithelial cells of the lung or intestine via a transport system mediated by the neonatal Fc receptor. The Fc domain conjugated peptide showed higher stability and absorbability in vivo than the wild type peptide. Heterodimer conjugates were found to exhibit even higher activity than single stranded forms.

Proteins and Peptides Various polypeptides and proteins can be used within the methods and compositions of the invention. In certain embodiments, the protein may comprise an antibody or antibody fragment comprising an antigen binding site. As used herein, a protein, polypeptide, or peptide is usually a protein having a number of amino acids greater than 200 and less than the full-length sequence translated from the gene, and a polypeptide having a number of amino acids greater than 100. And / or a peptide having about 3 to about 100 amino acids is referred to as, but not limited to, a peptide. For convenience, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably herein. Thus, the term “protein or peptide” includes amino acid sequences that include at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

  As used herein, “amino acid residue” means any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the protein residues or peptides are contiguous without non-amino acids inserted into the sequence of amino acid residues, and in other embodiments the sequence is one or more non-amino acid residues. May be included. In certain embodiments, one or more non-amino acid residues are inserted into the protein or peptide sequence.

Thus, the term “protein or peptide” includes an amino acid sequence comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, It is not limited to what is shown below.

  A protein or peptide is known to those skilled in the art by standard molecular biology techniques, such as expression of a protein, polypeptide or peptide, isolation of a protein or peptide from a natural product, or chemical synthesis of a protein or peptide. It is possible to manufacture using any method. Nucleotides corresponding to various genes, as well as proteins, polypeptides and peptide sequences have already been disclosed and can be found using computer databases known to those skilled in the art. One such database is the National Center for Biotechnology Information GenBank and the GenPept database (www.ncbi.nlm.nih.gov/). The coding domains of known genes can be amplified and / or expressed using the techniques described herein or techniques known to those skilled in the art. Alternatively, various commercially available proteins, polypeptides, and peptides are known to those skilled in the art.

In other embodiments relating to the method of preparation of a polypeptide, a peptidomimetic is used. Mimetics are molecules that contain peptides that mimic elements of protein secondary structure. See, for example, Johnson et al., “Peptide Turn Mimetics” in BIOTECHNOLOGYAND PHARMACY, edited by Pezzuto et al., Chapman and Hall, New York (1993). Each is incorporated herein by reference. The basis for using peptidomimetics is that protein peptide backbones, such as antibody and antigen peptide backbones, exist primarily to align amino acid side chains to promote molecular interactions. Peptidomimetics are expected to allow molecular interactions similar to natural molecules.

Fusion Protein Various embodiments relate to fusion proteins. These molecules usually have all or most of the peptide attached to all or part of the second polypeptide or protein at the N- or C-terminus. Methods for generating fusion proteins are well known to those skilled in the art. The protein encodes a second protein or peptide, for example, by chemical conjugation with a bifunctional crosslinker, by de novo synthesis of the entire fusion protein, or by a DNA sequence encoding the first protein or peptide. Can be produced by binding to the DNA sequence to be expressed and then expressing the entire fusion protein.

Synthetic peptides Proteins or peptides can be synthesized in whole or in part in solution or on a solid support using conventional techniques. Various automatic synthesizers are commercially available and can be used according to known procedures. For example, Stewart and Young, (1984, Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co.), Tam et al. (1983, J. Am. Chem. Soc, 105: 6442), Merrifield, (1986, Science 232: 341-347) and Barany and Merrifield (1979, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp. 1-284). Short peptide sequences, usually from about 6 up to 35-50 amino acids, can be easily synthesized using these methods. Alternatively, recombinant DNA technology can be used in which a nucleotide sequence encoding a peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate cell, and cultured under conditions suitable for expression. .

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を参照されたい。それぞれは、抗体分泌性ハイブリドーマ系列および抗体またはその断片に関連する抗原に対するATCC寄託番号に関して、参照により本明細書に援用される）。これらは単なる例示であり、他の広範な抗体分泌性ハイブリドーマが当技術分野では周知である。対象となる、選択された疾患に関連する標的に対する抗体に関する、ATCC、PubMed、および／または米国特許商標庁のデータベースを検索するだけで、ほとんどすべての疾患に関連する抗原に対する抗体分泌性ハイブリドーマが得られることを当業者であれば認識するであろう。 Antibodies Various embodiments relate to antibodies to the target. As used herein, the term “antibody” refers to any antibody-like molecule having an antigen binding site, Fab ′, Fab, F (ab ′) ₂ , single domain antibody (DAB), Fv And antibody fragments such as scFv (single chain Fv). Techniques for the preparation and use of various antibody-based compositions and fragments are well known in the art. Means for preparing and evaluating antibodies are also well known in the art (see, eg, Harlowe and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory). Useful antibodies are commercially available from a number of vendors. For example, various antibody-secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA, USA). A number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited with the ATCC and are available for use in the methods and compositions of the invention (see, eg, US Pat.

Please refer to the issue. Each of which is hereby incorporated by reference with respect to the ATCC deposit number for the antibody-secreting hybridoma line and antigen associated with the antibody or fragment thereof). These are merely examples, and a wide variety of other antibody-secreting hybridomas are well known in the art. Simply search the ATCC, PubMed, and / or U.S. Patent and Trademark Office databases for antibodies against the target of interest associated with the selected disease of interest to obtain antibody-secreting hybridomas against almost any disease-associated antigen. Those skilled in the art will recognize that

Production of Antibody Fragments Some embodiments relating to the methods and / or compositions of the invention relate to antibody fragments. The antibody fragment can be obtained by digesting the whole antibody with pepsin or papain using conventional methods. For example, antibody fragments can be produced by enzymatic degradation of antibodies using pepsin, resulting in F (ab ′) ₂ fragments. This fragment can be further broken down using a thiol reducing agent and then blocking the sulfhydryl groups that are optionally generated by cleavage of disulfide bonds. Alternatively, two monovalent Fab fragments and one Fc fragment are obtained by enzymatic degradation using papain n. For example, US Pat. No. 4,036,945, US Pat. No. 4,331,647, Nisonoff et al., 1960, Arch. Biochem. Biophys., 89: 230; Porter, 1959, Biochem. J., 73: 119, Edelman et al., 1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (Ed.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons). .

  As long as the fragment binds to the antigen recognized by the intact antibody, separation of the heavy chain to form a monovalent heavy-light chain fragment, further fragmentation of the fragment, or other enzymatic, chemical, or genetic Other methods of cleaving the antibody, such as a scientific technique, may be used. For example, the Fv fragment contains an association of VH and VL chains. This association may be non-covalent, as described by Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69: 2659. Alternatively, the variable domain chain may be bound by cross-linking with a compound such as an intermolecular disulfide bond or glutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech., 12: 437.

  Preferably, the Fv fragment has VH and VL chains connected by a peptide linker. These single chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL chains and connected by an oligonucleotide linker sequence. The structural gene is inserted into an expression vector and then introduced into a suitable cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide that bridges the two variable domains. Methods for producing sFv are well known in the art. Whitlow et al., 1991, Methods: A Companion to Methods in Enzymology 2:97, Bird et al., 1988, Science, 242: 423, U.S. Pat.No. 4,946,778, Pack et al., 1993, Bio / Technology, 11: 1271, and Sandhu, 1992, Crit. Rev. Biotech., 12: 437.

  Another form of antibody fragment is peptide coding for a single complementarity determining domain (CDR). CDR peptides ("minimal recognition units") are obtained by constructing a gene that encodes the CDR of the antibody of interest. The gene is obtained, for example, by synthesizing a variable domain derived from RNA of an antibody-producing cell using a polymerase chain reaction. Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2: 106; Ritter et al. (Ed.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (Cambridge University Press), Birch et al. ( Ed.), 1995, MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.). When the antibody-secreting hybridoma series is published, a CDR sequence encoding the antigen-antibody specificity can be obtained, introduced into a chimeric antibody or a humanized antibody, and used.

Chimeric and humanized antibodies A chimeric antibody is one in which the variable domain of a human antibody is replaced by a murine variable domain, such as the murine complementarity determining domain (CDR). Chimeric antibodies exhibit low immunogenicity and high stability when administered to a subject. Methods for constructing chimeric antibodies are well known in the art (eg, Leung et al., 1994, Hybridoma 13: 469).

  A chimeric monoclonal antibody can be humanized by introducing murine immunoglobulin heavy and light chain variable domain mouse CDRs into the corresponding variable domains of a human antibody. The mouse framework domain (FR) in the chimeric monoclonal antibody is also replaced with a human FR sequence. In order to preserve the stability and antigen specificity of the humanized monoclonal antibody, one or more human FR residues can be replaced with the corresponding residues in mouse. Humanized monoclonal antibodies can be used to treat patients. By selectively modifying the CDR sequences, the affinity of the humanized antibody for the target can also be increased (WO 00/29584). Techniques for producing humanized monoclonal antibodies are well known in the art (eg, Jones et al., 1986, Nature, 321: 522, Riechmann et al., Nature, 1988, 332: 323, Verhoeyen et al., 1988, Science , 239: 1534, Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89: 4285, Sandhu, Crit. Rev. Biotech., 1992, 12: 437, Tempest et al., 1991, Biotechnology 9: 266, See Singer et al., J. Immunol., 1993, 150: 2844).

  Other embodiments relate to non-human primate antibodies. General techniques for obtaining therapeutically useful antibodies from baboons are described, for example, in Goldenberg et al., WO 91/11465 (1991), and Losman et al., Int. J. Cancer 46: 310 (1990). Has been.

Human antibodies Combinatorial methods or methods for producing fully human antibodies using transgenic animals transformed with human immunoglobulin loci are well known in the art (eg, Mancini et al., 2004, New Microbiol. 27: 315-28; Conrad and Scheller, 2005, Comb. Chem. High Throughput Screen. 8: 117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol. Incorporated herein by reference). The fully human antibody has lower side effects than a chimeric antibody or a humanized antibody, and is expected to basically function as a human antibody in vivo. In certain embodiments, the methods and procedures of the invention can be used on human antibodies produced by the techniques.

  In some variations, phage display methods can be used to produce humanized antibodies (see, eg, Dantas-Barbosa et al., 2005, Genet. MoI. Res. 4: 126-40, the contents of which are Incorporated herein by reference). Humanized antibodies can be generated from normal humans or from humans that exhibit certain pathological conditions such as cancer (Dantas-Barbosa et al., 2005). An advantage of constructing human antibodies from diseased humans is that the humoral antibody repertoire can be biased towards antibodies against antigens associated with the disease.

  In one non-limiting example of this method, Dantes-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Usually, total RNA was obtained from circulating lymphocytes (Id). Recombinant Fab was cloned from μ, γ, and K chain antibody repertoires and inserted into a phage display library (Id). RNA was converted to cDNA and used to create a Fab cDNA library using primers specific for immunoglobulin heavy and light chain sequences (Marks et al., 1991, J. MoI. Biol. 222: 581). -97, the contents of which are incorporated herein by reference). The library was constructed according to the method of Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (Ed.), 1st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 9.1 to 9.22, the contents of which are incorporated herein by reference). The finally obtained Fab fragment was digested with restriction endonucleases and inserted into the bacteriophage genome to create a phage display library. As is well known in the art, the library can be screened by conventional phage display methods. One skilled in the art will recognize that this approach is exemplary only and that any known method for producing and screening human antibodies by phage display methods can be used.

  In other variations, transgenic animals genetically modified to produce human antibodies can be used to produce antibodies against almost any immune target using normal immunization procedures. A non-limiting example of such a system is the XenoMouse® from Abgenix (Fremont, CA, USA) (see, eg, Green et al., 1999, J. Immunol. Methods 231: 11-23). The contents of which are incorporated herein by reference). In XenoMouse® and similar animals, the immune system of other mice remains intact, but the mouse antibody gene is inactivated and functionally replaced with a human antibody gene.

  Transformation of The XenoMouse® with a germline constructed YAC (yeast artificial chromosome) containing part of the human IgH and Igκ loci, including most of the variable domains, along with accessory and regulatory genes did. The human variable domain repertoire can be used to generate B cells having antibody-producing ability, and can also be made into a hybridoma using a known technique. XenoMouse® immunized with a target antigen produces human antibodies by a normal immune response, but can be recovered and / or manufactured using the normal techniques described above. Various variants can be obtained for the XenoMouse®, each producing a different class of antibody. The human antibody can be conjugated to other molecules by chemical cross-linking or other known methods. Human antibodies produced by transgenic methods have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). One skilled in the art will recognize that the compositions and methods of the present invention are not limited to the XenoMouse® system, and that any transgenic animal modified to produce human antibodies can be used. Let's go.

Pre-targeting One strategy for using a bispecific bioactive assembly is a pre-targeting method in which the effector molecule is administered after the bispecific assembly is administered. The bispecific assembly includes a binding site for an effector, hapten, or carrier and is localized to the diseased tissue to improve the specificity of the localization of the effector to the diseased tissue (US Patent Publication No. 2005002945). issue). Since effector molecules are cleared from the circulatory system much faster than bispecific assemblies, when using pre-targeting methods, contact between normal tissue and the effector molecule is an antibody that targets the effector molecule to the disease. It is less than the case where it is directly bonded to.

  Pre-targeting methods have been developed to increase the target to background ratio in detection or therapeutic agents. For examples of pre-targeting methods and approaches with biotin / avidin ratio, see, for example, Goodwin et al., US Pat. No. 4,863,713, Goodwin et al., J. Nucl. Med. 29: 226, 1988, Hnatowich et al., J Nucl. Med. 28: 1294, 1987, Oehr et al., J. Nucl. Med. 29: 728, 1988, Klibanov et al., J. Nucl. Med. 29: 1951, 1988, Sinitsyn et al., J. Nucl. Med. 30:66, 1989, Kalofonos et al., J. Nucl. Med. 31: 1791, 1990, Schechter et al., Int. J. Cancer 48: 167, 1991, Paganelli et al., Cancer Res. 51: 5960, 1991, Paganelli et al., Nucl. Med. Commun. 12: 211, 1991, US Pat. No. 5,256,395, Stickney et al., Cancer Res. 51: 6650, 1991, Yuan et al., Cancer Res. 51: 3119, 1991, US Pat. , 077,499, U.S. Patent Application No. 09 / 597,580, U.S. Patent Application No. 10 / 361,026, U.S. Patent Application No. 09 / 337,756, U.S. Patent Application No. 09 / 823,746, US Patent Application No. 1 / 116,116, U.S. Patent Application No. 09 / 382,186, U.S. Patent Application No. 10 / 150,654, U.S. Patent No. 6,090,381, U.S. Patent No. 6,472,511, U.S. Patent Application No. 10 / 114,315, U.S. Provisional Patent Application No. 60 / 386,411, U.S. Provisional Patent Application No. 60 / 345,641, U.S. Provisional Patent Application No. 60 / 3328,835, U.S. Provisional Patent Application No. See U.S. Patent Application No. 60 / 426,379, U.S. Patent Application No. 09 / 823,746, U.S. Patent Application No. 09 / 337,756, and U.S. Provisional Patent Application No. 60 / 342,103. Each document is incorporated herein by reference.

  In certain embodiments, bispecific assemblies and targetable constructs are described, for example, in US Pat. Nos. 6,126,916, 6,077,499, 6,010,680, 5,776, 095, 5,776,094, 5,776,093, 5,772,981, 5,753,206, 5,746,996, 5,697,902 , 5,328,679, 5,128,119, 5,101,827, and 4,735,210, and target and / or imaging of normal or diseased tissue Useful for. Other methods are described in US application Ser. No. 09 / 337,756, filed Jun. 22, 1999, and U.S. patent application Ser. No. 09 / 823,746, filed Apr. 3, 2001. The contents of each are incorporated herein by reference.

Aptamer In certain embodiments, the precursor of bioactive assembly formation may include an aptamer. Methods for aptamer construction and evaluation of binding properties are well known in the art. For example, such techniques are described in US Pat. Nos. 5,582,981, 5,595,877, and 5,637,459. Each is incorporated herein by reference.

  Aptamers can be prepared using any known method, such as synthetic, recombinant, and purification methods, and can be used alone or with other ligands that have specificity for the same target. In general, a minimum of about 3 nucleotides, preferably 5 or more nucleotides are required to cause specific binding. Aptamers consisting of 10, 20, 30, or 40 nucleotides are preferred, but aptamers with sequences shorter than 10 bases are also possible.

  Aptamers need to contain sequences that exhibit binding specificity, but may be bound to flanking domains and other derivatives. In a preferred embodiment, the aptamer binding sequence can be flanked by primer binding sequences to facilitate the amplification of the aptamer by PCR or other amplification methods. In other embodiments, the flanking sequence may include a specific sequence that preferentially recognizes or binds residues to facilitate immobilization of the aptamer to the substrate.

Aptamers can be isolated, sequenced, and / or amplified or synthesized in the same way as normal DNA or RNA molecules. Alternatively, the subject aptamer may contain a modified oligomer. The hydroxyl groups normally present in aptamers can be activated to replace phosphonic acid groups, phosphate groups, protect with normal protecting groups, or further form bonds to other nucleotides, Can be combined. P (O) O substituted with one or more phosphodiester bonds with P (O) S, P (O) NR ₂ , P (O) R, P (O) OR ¹ , CO, or CNR ₂ Wherein R is H or alkyl (1-20C) and R ¹ is alkyl (1-20C), and can be substituted with other linking groups such as O or S Can be linked to adjacent nucleotides. The bonds in the oligomer need not all be the same.

  Methods for preparing and screening aptamers that bind to a particular target of interest are well known, see, eg, US Pat. No. 5,475,096 and US Pat. No. 5,270,163. Each is incorporated herein by reference. Techniques typically involve selecting from a mixture of candidate aptamers, repeating the binding stepwise, separating bound aptamers from unbound, and amplifying. Since there are only a few sequences in the mixture that correspond to the aptamers with the highest affinity (possibly only one molecule of aptamer), a sufficient amount of aptamers in the mixture (about 5-50). %) Is usually desirable to ensure that it remains after separation. Each cycle enriches aptamers with high affinity for the target. To purify aptamers with high affinity and specificity for the target, the selection and enrichment cycle can be repeated 3-6 times.

Avimer In certain embodiments, the peripheral modules and / or assemblies described herein may include one or more avimer sequences. Avimers are a type of binding protein that is somewhat similar to antibodies in affinity and specificity for various targets. These were developed from human extracellular receptor domains by in vitro exon shuffling and phage display (Silverman et al., 2005, Nat. Biotechnol. 23: 1493-94, Silverman et al., 2006, Nat. Biotechnol. 24: 220). The resulting multidomain protein may contain multiple independent binding domains that exhibit higher affinity (may be subnanomolar) and specificity than binding proteins with a single epitope. (Id). In various embodiments, the avimer may be associated with a DDD and / or AD sequence, eg, for use in the methods and compositions of the invention. Detailed methods for the construction and use of avimers are disclosed, for example, in U.S. Patent Application Publication Nos. 20040175756, 20050048512, 20050053973, 20050089932, and 2005021384, each example being incorporated by reference. Incorporated herein by reference.

In Vitro Diagnosis Based on Protein Detection, Diagnosis, and Imaging Methods The present invention is directed to the use of bioactive assemblies for screening biological samples for the presence of disease-related antigens in vitro and / or in vivo. And In a typical immunoassay, a bioactive assembly comprising an antibody, fusion protein, or fragment thereof can be used in the liquid phase or bound to a solid support as described below, and in preferred embodiments, in particular Administered in vivo, the antibody or fragment thereof is humanized. It is also preferred that the antibody or fragment thereof is a fully human antibody or fragment thereof. Even more preferably, the fusion protein comprises a humanized or fully human antibody. A wide range of techniques are known for determining the expression level of a particular gene and any known method such as immunoassay, RT-PCR, mRNA purification, and / or cDNA preparation and hybridization to a gene expression analysis chip One skilled in the art will recognize that can be used to determine expression levels in individual subjects and / or tissues. Typical examples of useful in vitro analyzes include RIA, ELISA, sandwich ELISA, Western blot, slot blot, dot blot, and the like. The technique was developed using intact antibodies, but bioactive assemblies into which antibodies, antibody fragments or other binding residues have been introduced can be used.

  Bioactive assemblies incorporating antibodies, fusion proteins, antibody fragments, and / or other binding residues can be used for detection of target antigens in tissue sections prepared from histological specimens. The in situ detection can be used to determine the presence of the antigen and to determine the distribution of the antigen in the specimen tissue. In situ detection can be performed by applying an assembly with a detectable label to a frozen or paraffin-embedded tissue section. General techniques of in situ detection are well known to those skilled in the art. See, for example, Ponder, “Cell Marking Techniques and Their Application” in MAMMALIAN DEVELOPMENT: A PRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987) and Coligan pages 5.8.1-5.8.8.

  For example, using any suitable marker residue such as a radioisotope, an enzyme, a fluorescent label, a dye, a chromophore, a chemiluminescent label, a bioluminescent label, or a paramagnetic label, a detectable label is attached to the bioactive assembly. It can be carried out.

  The marker residue may be a radioisotope detected by such means as a gamma dosimeter, or a beta scintillation counter, or autoradiography. In preferred embodiments, the diagnostic conjugate is a gamma ray, a beta ray, or a positron emitting isotope. A marker residue refers to a single molecule that generates a signal under certain conditions. Examples of marker residues include radioisotopes, enzymes, fluorescent labels, dyes, chromophores, chemiluminescent labels, bioluminescent labels, and paramagnetic labels. The binding of the marker residue to the bioactive assembly can be done using standard methods well known in the art. The usual methods for this are Kennedy et al., Clin. Chim. Acta 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1 (1977), Shih et al., Infl J. Cancer 46: 1101 (1990). It is described in.

In Vivo Diagnostics Diagnostic imaging with labeled peptides or MAbs is well known. For example, in immunoscintigraphy, a ligand or antibody is labeled with a γ-radioactive radioisotope and introduced into the patient's body. Using a γ-ray camera, the position and distribution of γ-ray radioactive isotopes are detected. For example, Chase from Srivastava (edition), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press 1988), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (Edition), pp. 624-652 (Mack Publishing Co., 1990) See "Medical Applications of Radioisotopes" and Brown, "Clinical Use of Monoclonal Antibodies", BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al., (Ed.) (Chapman & Hall 1993). ^{18 F, 68 Ga, 64 Cu} , and the like ¹² 4I, such as those having an energy of 511 keV, it is also preferable to use a positron-emitting radioisotopes (PET isotopes). The imaging can be performed by direct labeling of the bioactive assembly or by the pre-target imaging method described by Goldenberg et al., “Antibody Pre-targeting Advances Cancer Radioimmunodetection and Radioimmunotherapy” (J Clin Oncol 2006; 24: 823-834). it can. See also U.S. Patent Application Publication Nos. 20050002945, 20040018557, 20030148409, and 2005014207. Each is incorporated herein by reference.

The radiation dose delivered to the patient is kept as low as possible by choosing the isotope for the best combination of minimum half-life, minimum body retention time, and minimum amount that can be detected and accurately measured . Suitable radioisotopes for diagnostic imaging include ^99m Tc and ¹¹¹ In.

  Bioactive assemblies, or haptens or carriers that bind to them, can also be labeled with paramagnetic ions and various radiocontrast agents for in vivo diagnosis. Particularly useful contrast agents for magnetic resonance imaging include gadolinium, manganese, dysprosium, lanthanum, or iron ions. Other contrast agents include chromium, copper, cobalt, nickel, rhenium, europium, terbium, holmium, or neodymium. Ligands, antibodies and fragments thereof can also be conjugated with ultrasound contrast / enhancement agents. For example, one ultrasound contrast agent is a liposome containing humanized IgG or a fragment thereof. It is also preferred that the ultrasound imaging agent is a gas-filled liposome.

Imaging Agents and Radioisotopes Many suitable imaging agents are known with methods for binding them to proteins or peptides (see, eg, US Pat. Nos. 5,021,236 and 4,472,509). Both of which are incorporated herein by reference). One conjugation method involves the use of metal chelate complexes using, for example, organic chelating agents such as DTPA bound to proteins or peptides (US Pat. No. 4,472,509). The protein or peptide may be reacted with the enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers can be prepared in the presence of these coupling agents or by reaction with iothiocyanate.

  Non-limiting examples of paramagnetic ions that could potentially be used include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), Copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III) Gadolinium is particularly preferable. Ions useful in other situations such as X-ray imaging include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III).

Examples of radioisotopes that can be used as imaging agents or therapeutic agents include astatine, carbon ¹⁴ , chromium ⁵¹ , chlorine ³⁶ , cobalt ⁵⁷ , cobalt ⁵⁸ , copper ⁶² , copper ⁶⁴ , copper ⁶⁷ , Eu ¹⁵² , fluorine ¹⁸ , gallium ⁶⁷ , gallium ⁶⁸ , hydrogen ³ , iodine ¹²³ , iodine ¹²⁴ , iodine ¹²⁵ , iodine ¹³¹ , indium ¹¹¹ , iron ⁵² , iron ⁵⁹ , lutetium ¹⁷⁷ , phosphorus ³² , phosphorus ³³ , rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , Sc ⁴⁷ , Selenium ⁷⁵ , silver ¹¹¹ , sulfur ³⁵ , technetium ^94m , technetium ^99m , yttrium ⁸⁶ , yttrium ⁹⁰ , and zirconium ⁸⁹ may be mentioned. I ¹²⁵ is often preferred for use in certain embodiments, and technetium ^99m and indium ¹¹¹ are also often preferred because of their low energy and suitability for long-term detection.

Radiolabeled proteins or peptides can be produced using methods well known in the art. For example, they can be iodinated by contacting them with a chemical oxidant such as sodium or potassium iodide and sodium hypochlorite, or an enzyme oxidant such as lactoperoxidase. The protein or peptide can be obtained, for example, by reducing pertechnetate with a tin (II) solution, chelating the reduced technetium on a Sephadex column and passing the peptide through this column, or by, for example, pertechnetate. It can be labeled with technetium ^99m by a direct labeling method in which a reducing agent such as SnCl ₂ , a buffer solution such as sodium potassium phthalate solution, and a peptide are incubated. Intermediate functional groups often used to bind radioisotopes present as metal ions to peptides include diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, porphyrin chelating agent, and ethylenediaminetetraacetic acid (EDTA). Can be mentioned. The use of fluorescent labels such as rhodamine, fluorescein isothiocyanate is also contemplated.

  In certain embodiments, the protein or peptide may be bound to a secondary binding ligand or enzyme (such as an enzyme tag) that produces a colored product upon contact with the chromogenic material. Suitable enzymes include urease, alkaline phosphatase, (horseradish) hydroperoxidase, and glucose oxidase. Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those skilled in the art, for example, U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, , 277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference. These fluorescent labels are preferred for in vitro use, but are also useful for in vivo applications, particularly in endoscopic or intravascular detection methods.

  In other embodiments, the ligand, antibody, or other protein or peptide may be labeled with a fluorescent marker. Non-limiting examples of photodetectable labels include Alexa 350, Alexa 430, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX 5-carboxy-4 ′, 5′-dichloro-2 ′, 7′-dimethoxyfluorescein, 5-carboxy-2 ′, 4 ′, 5 ′, 7′-tetrachloro-dimethoxyfluorescein, 5-carboxyfluorescein, 5 -Carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylamino, cascade blue, Cy2, Cy3, Cy5, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa- 1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, tele Taric acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptate complex, europium trisbipyridinediamine, Europium cryptate or chelate complex, diamine, dicyanine, La Jolla blue dye, allopicocyanine, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythocyanin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine Edans, and include Texas Red. These and other luminescent labels are available from commercial sources such as Molecular Probes (Eugene, Oreg., USA), and EMD Biosciences (San Diego, CA, USA).

  Useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters, or bioluminescent compounds such as luciferin, luciferase, and aequorin. The diagnostic conjugate can be used, for example, for intraoperative, endoscopic, or intravascular diagnosis of a tumor or disease.

  In various embodiments, useful labels may include metal nanoparticles. Methods for preparing nanoparticles are known (eg, US Pat. Nos. 6,054,495, 6,127,120, 6,149,868, Lee and Meisel, J. Phys. Chem. 86: 3391-3395, 1982). Nanoparticles are also commercially available from commercial sources (eg Nanoprobes Inc., Yabank, NY, USA, Polysciences, Inc., Warrington, PA, USA). Modified nanoparticles such as Nanogold (R) commercially available from Nanoprobes Inc. (Yabank, NY, USA) are commercially available. Functionalized nanoparticles useful for conjugation with proteins or peptides are commercially available.

Therapeutic Drug Pharmaceutical Compositions In certain embodiments, a bioactive assembly and / or one or more therapeutic agents can be administered to a subject, such as a subject with cancer. The agent can be administered in the form of a pharmaceutical composition. This usually involves the preparation of a composition that is substantially free of impurities that are harmful to humans or animals. It is known to those skilled in the art that the pharmaceutical composition can be administered to a subject by various routes such as oral or parenteral such as intravascular.

  In certain embodiments, an effective amount of a therapeutic agent must be administered to the subject. An “effective amount” is the amount of drug that will produce the desired effect. The effective amount depends, for example, on the effectiveness of the drug and the intended effect. For example, for the treatment of hyperplastic symptoms such as macular degeneration or endometriosis, anti-angiogenesis compared to the treatment of cancer aimed at reducing or eliminating solid cancer or preventing or reducing metastasis. It is necessary to reduce the dose of the drug. An effective amount of a particular agent for a particular purpose can be determined using methods well known to those skilled in the art.

Chemotherapeutic Agents In certain embodiments, chemotherapeutic agents can be administered. Examples of chemotherapeutic agents having anticancer activity include 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agent, etoposide (VP16) farnesyl-protein transferase inhibitor, gemcitabine, ifosfamide, mechloretamine, melphalan, methotrexate, mitomycin, navelbine, nitrosourea, pricomomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (DTIC aqueous solution), trans Platinum, vinblastine and methotrexate, vincristine, or any analog of the above Or a derivative | guide_body variant is mentioned, However, It is not limited to these. Chemotherapeutic agents useful for infectious microorganisms include acyclovir, albendazole, amantadine, amikacin, amoxicillin, amphotericin B, ampicillin, aztreonam, azithromycin, bacitracin, bactrim, batrafen (registered trademark), bifonazole carbenicillin, caspofandine , Cefaclor, cefazolin, cephalosporin, cefepime, ceftriaxone, cefotaxime, chloramphenicol, cidofovir, cypro®, clarithromycin, clavulanic acid, clotrimazole, cloxacillin, doxycycline, econazole, erythrocycline , Erythromycin, furadyl, fluconazole, flucytosine, foscanet, furazolidone, ganciclovir, gentamicin , Imipenem, isoniazid, itraconazole, kanamycin, ketoconazole, lincomycin, linezolid, meropenem, miconazole, minocycline, naphthifine, nalidixic acid, neomycin, netilmicin, nitrofurantoin, nystatin, oseltamivir, oxacillin, penimorazoline penicarapine , Rifabutin, rifampin rimantadine, streptomycin, sulfamethoxazole, sulfasalazine, tetracycline, thioconazole, tobramycin, tolcyclate, tolnaftate, trimethoprim, sulfamethoxazole, valaciclovir, vancomycin, zanamil, and dysomycin. It is not limited.

  Chemotherapeutic agents and administration methods, dosages, etc. are well known to those skilled in the art (see, for example, “Physicians Desk Reference”, “The Pharmacological Basis of Therapeutics” and “Remington's Pharmaceutical Sciences” by Goodman & Gilman. The contents are incorporated herein by reference to the relevant parts). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any case, determine the appropriate dose for the individual subject.

Hormones Corticosteroid hormones are often used in combination therapy because they can increase the effectiveness of other chemotherapeutic agents. Predomison and dexamethasone are examples of corticosteroid hormones. Progestins such as hydroxyprogesterone caproate, methoxyprogesterone, and megestrol acetate are used for endometrial and breast cancer. Estrogens such as diethylstilbestrol and ethinyl estradiol are used for cancers such as prostate cancer. Antiestrogens such as tamoxifen are used for cancers such as breast cancer. Androgens such as testosterone propionate and fluoxymesterone are also used to treat breast cancer.

Angiogenesis inhibitors In certain embodiments, angiogenesis inhibitors such as angiostatin, baculostatin, canstatin, maspin, anti-VEGF, antibodies, anti-PIGF peptides and antibodies, anti-angiogenic growth factors, anti-Flk-1 antibodies, Anti-Flt-1 antibody and peptide, laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxy Estradiol, growth-related protein, carboxamidotriazole, CM101, marimastat, pentosan polysulfate, angiopoietin-2, interferon α, herbimycin A, PNU145156E, 16K prolactin fragment, Amide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, actin (accutin), angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline, is useful.

Immunomodulator As used herein, the term “immunomodulator” includes interleukins, colony stimulating factors, interferons (such as interferons α, β, and γ), and stem cell growth factors called “S1 factors”, etc. Cytokines, stem cell growth factors, lymphotoxins, and hematopoietic factors. Is included. Suitable immunomodulator residues include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferon γ, TNF-α.

  The term “cytokine” is a generic term for proteins or peptides that are released from a cell population and act as intercellular transmitters in other cells. As used broadly herein, examples of cytokines include lymphokines, monokines, growth factors, and conventional polypeptide hormones. Cytokines include human growth hormone, growth hormone such as N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH). Glycoprotein hormones such as luteinizing hormone (LH), liver growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, tumor necrosis factor-α and -β, Muellerian tube inhibitor, Peptides related to mouse gonadotropin, inhibitor, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor such as NGF-β, transforming growth factor such as platelet growth factor, TGF-α and TGF-β ( TG ), Insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factor, interferons such as interferon α, β, and γ, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-) CSF), colony stimulating factor (CSF) such as 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 Such as interleukin (IL), LIF, G-CSF, GM-CSF, M-CSF3, EPO, kit-ligand or FLT-3, angiostatin, thrombospondin, endos Chin, is one of tumor necrosis factor, and LT. As used herein, the term cytokine includes those derived from naturally occurring or recombinant cell lines, and those that exhibit the same physiological activity as native sequence cytokines.

  Chemokines usually act as chemoattractants that collect immune effector cells at chemokine expression sites. Chemokines include, but are not limited to, RANTES, MCAF, MIP1-α, MIP1-β, and IP-10. One skilled in the art will recognize that certain cytokines are known to have chemoattractive effects and can also be classified as chemokines. Similarly, the terms immunomodulator and cytokine overlap with each other.

Radiotherapy and radioimmunotherapy In certain embodiments, the peptides and / or proteins are useful for radionuclide therapy or radioimmunotherapy (see, eg, Govindan et al., 2005, Technology in Cancer Research & Treatment, 4: 375-91, See Sharkey and Goldenberg, 2005, J. Nucl. Med. 46: 115S-127S, Goldenberg et al. (J Clin Oncol 2006; 24: 823-834), “Antibody Pre-targeting Advances Cancer Radioimmunodetection and Radioimmunotherapy”. Each of which is incorporated herein by reference). In a specific embodiment, the bioactive assembly can be directly labeled with a useful radioisotope and administered to a subject. In another embodiment, a haptenic peptide or ligand that is labeled with a radioisotope and injected after administration of a bispecific bioactive assembly that is localized to a site of increased expression in a diseased tissue In use, radioisotopes can be administered by the pre-target method described above.

Radioisotopes useful for the treatment of diseased tissue include ¹¹¹ In, ¹⁷⁷ Lu, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ⁶² Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ³² P, ³³ P, ⁴⁷ Sc, ¹¹¹ Ag, ⁶⁷ Ga, ¹⁴² Pr, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, ²¹² Pb, ²²³ Ra, ²²⁵ Ac, ⁵⁹ Fe, ⁷⁵ Se, ⁷⁷ As, ⁸⁹ Sr, ⁹⁹ Mo, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹⁴³ Pr, ¹⁴⁹ Pm, ¹⁶⁹ Er, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, and ²¹¹ Pb. The therapeutic radioisotope is preferably 20 to 6,000 keV, the Auger electron emitter is preferably 60 to 200 keV, the β emitter is preferably 100 to 2,500 keV, and the α emitter is used. , Preferably having a decay energy of 4,000 to 6,000 keV. The maximum decay energy of useful beta particle emitting nuclei is preferably 20 to 5,000 keV, more preferably 100 to 4,000 keV, most preferably 500 to 2,500 keV. Also preferred are radionuclides that decay almost entirely with the emission of Auger electrons. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m, and Ir-192 It is. Useful beta particle emitting nuclides preferably have a decay energy of <1,000 keV, more preferably <100 keV, and most preferably <70 keV. Also preferred are radionuclides that decay almost entirely with the release of alpha particles. Examples of the radionuclide include Dy-152, At-2115, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213. , And Fm-255, but are not limited to these. The decay energy of useful alpha particle emitting nuclides is preferably 2,000 to 10,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV.

For example, ⁶⁷ Cu, which has a half-life of 61.5 hours and is a natural source of beta particles and gamma rays, is considered one of the more promising radioisotopes in radioimmunotherapy. The chelating agent p-bromoacetamidobenzyltetraetheramine tetraacetic acid (TETA) can be used to conjugate to proteins or peptides. Alternatively, ⁹⁰ Y that releases high energy β particles can be bound to peptides, antibodies, fusion proteins, or fragments thereof using diethylenetriaminepentaacetic acid (DTPA).

Other possible radioisotopes include ¹¹ C, ¹³ N, ¹⁵ O, ⁷⁵ Br, ¹⁹⁸ Au, ²²⁴ Ac, ¹²⁶ I, ¹³³ I, ⁷⁷ Br, ^{113 m} In, ⁹⁵ Ru, ⁹⁷ Ru, ¹⁰³ Ru. ^{, 105 Ru, 107 Hg, 203} Hg, 121m Te, 122m Te, 125m Te, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 105 Rh, 142 Pr, 143 Pr, 161 Tb, 166 Ho, 199 Au, ⁵⁷ Co, ⁵⁸ Co, ⁵¹ Cr, ⁵⁹ Fe, ⁷⁵ Se, ²⁰¹ Tl, ²²⁵ Ac, ⁷⁶ Br, ¹⁶⁹ Yb and the like.

  In other embodiments, radiosensitizers can be used. The addition of a radio sensitizer can increase efficiency. Radiosensitizers are described in D. M. Goldenberg (eds.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRC Press (1995), the entire contents of which are hereby incorporated by reference.

  Peptides, antibodies, antibody fragments, or fusion proteins with carriers that contain boron addend-loaded for thermal neutron beam activation therapy work similarly. However, it is advantageous to wait for the immunoconjugate not bound to the target to be discharged before performing the thermal neutron irradiation. Using an antibody that binds to the ligand can expedite elimination. See U.S. Pat. No. 4,624,846 for a description of the general principles. For example, for example, a boron adduct such as carborane can be bound to the antibody. As is well known in the art, carboranes having a carboxyl group in the pendant side chain can be prepared. Binding of carborane to a carrier such as aminodextran can be performed by activating the carboxyl group of carborane and condensing with an amine on the carrier. The intermediate conjugate is then conjugated with an antibody. After administration of the conjugate, the boron adduct is activated by thermal neutron irradiation and is converted to a radioactive atomic species that is highly toxic and has a short range effect by alpha decay.

Kits Various embodiments may relate to kits that include components suitable for the treatment or diagnosis of a patient's diseased tissue. A typical kit includes at least one bioactive assembly. If the composition comprising the administration component is not formulated for delivery via the gastrointestinal tract, such as oral administration, it may be accompanied by a device that can deliver the kit component by some other route. One type of device for use in parenteral administration is a syringe for injecting the composition into the body of a subject. An inhaler can also be used.

  The kit components may be stored together or may be stored separately in two or more separate containers. In certain embodiments, the container may be a vial containing a formulation of a sterile, lyophilized composition suitable for reconstitution. The kit may contain one or more buffer solutions suitable for reconstitution and / or dilution of other drugs. Other containers that can be used include, but are not limited to, pouches, trays, boxes, tubes, and the like. The components of the kit are stored in a container and kept in a sterilized state. Another element that can be included is a usage note for the user.

The following examples are given for illustration, but do not limit the scope of the claims of the present invention.
Example 1 Generation and Use of a Fusion Protein (Type a Adapter Module) Containing Heat Shock Protein, AD2, and AD3

A fusion protein in which AD2 and AD3 are attached to the amino and carboxyl termini of a heat shock protein such as HSP70 or gp96, respectively, includes a homodimer (X ₂ ) attached to DDD2 and the other to the DDD3C. _Two peripheral modules containing Y ₂ ) can be locked after docking to form a complex consisting of X ₂ (Ma) ₂ Y ₂ . One of the choices for the two peripheral modules is the human CD22 Ig-like domains 1 and 2 and the extracellular domain of human CD20, which when combined with an adapter module based on HSP is useful as a therapeutic vaccine for B-cell lymphoma It is. Another choice for the two peripheral modules is the CEACAM5 N-A1 and A3-B domains, and when conjugated with an adapter module based on HSP, is useful as a therapeutic vaccine for CEA-expressing cancers. The two peripheral modules may be the hLL1 Fab domain and the extracellular domain of HER2, and when conjugated with an adapter module based on HSP is useful as a therapeutic vaccine against cancers that overexpress HER2.

  As described above, a vaccine comprising AD2-HSP70-AD3 stably bound to the N-A1 and A3-B domains of CEACAM5 was prepared and after dispensing in saline or other physiologically acceptable solution, the colorectal colon Administered to patients after cancer resection. The therapeutic vaccine was administered once a week for a minimum of 4 weeks at a dose of 100-5000 μg, with a preferred dose of 500 μg. The injection route was subcutaneous, but the location of injection was changed each time, and injections to the same location were repeated at one or more intervals. For example, the first injection is the left thigh, the second injection is the right thigh, the third injection is the left arm, the fourth injection is the right arm, the fifth injection is the left thigh, the sixth injection Was the right arm, etc. After the end of the first cycle of 4 weeks of injection, two more weekly injections were given, followed by monthly injections. The effect of the vaccine on the induction of an anti-cancer immune response is: (1) delayed hypersensitivity as an assessment of cellular immunity, (2) in vitro activity of cytolytic T cells, (3) levels of humoral CEA, (4) Changes in tumor size using an imaging method such as CT scan, and (5) measurement by measurement of other biomarkers related to CEA-expressing cancer.

Example 2 Treatment of colorectal cancer with a vaccine comprising AD2-HSP70-AD3 stably linked to the N-A1 and A3-B domains of CEACAM5 Patient DN was 62 years old who had undergone resection of left colon cancer 4 cm in diameter A man was diagnosed with T2N1M0, refused postoperative chemotherapy and received an experimental vaccine. The patient received a vaccine containing a saline solution of AD2-HSP70-AD3 stably bound to the N-A1 and A3-B domains of CEACAM5 at a dose of 500 μg for 4 weeks, followed by 2 injections of the same dose twice a week. Thereafter, the same dose was administered monthly. The first injection was received in the left arm, the second time in the right arm, the third time in the right thigh and the fourth time in the left thigh. Thereafter, injections at these positions were repeated. To alleviate the side effects, the patient received pre-administration of Tylenol and antihistamines.

  During administration, grade 1 or 2 erythema and pruritus at the injection site were recorded as shortness of breath after the fourth dose, but all disappeared within 4 hours. After 3, 6 and 12 months, follow-up studies such as diagnostic imaging test (CT and FDG-PET test once after 12 months) and serum CEA analysis were performed, but no abnormality was detected. In a follow-up study two years later, the patient was not afflicted with the disease, and the administration of this experimental vaccine seems to have avoided the side effects of aggressive chemotherapy.

Example 3 Generation and Use of DDD2-hP1-DDD3C (Type b Adapter Module) Polypeptides fused to DDD2 and DDD3C at the amino and carboxyl termini of hP1, respectively, self-associate and bind via disulfide bonds Of hP1 homodimer and reduced with a reagent containing thiol, one containing a substance (X) derivatized with AD2 and the other two containing a substance (Y) derivatized with AD3 The module is further docked and fixed to form a complex consisting of X (hP1) ₂ Y. Suitable X and Y choices include receptor binding ligands, antibody fragments, and immunostimulatory molecules. For example, an X (hP1) ₂ Y construct, one of two peripheral modules based on an anti-hTfR (human transferrin receptor) Fab domain and the other based on an anti-hIR (human insulin receptor) Fab domain, For treatment, it can be used to deliver a therapeutic siRNA or gene across the blood brain barrier (BBB) and further to glioma cells (Zhang et al., Clin Cancer Res, 2004, 10: 3667-3677). ).

Example 4 Production and Use of DDD3-hP1-AD2 (Type c Adapter Module) A polypeptide in which DDD3 and AD2 are fused to the amino and carboxyl termini of hP1, respectively, is self-assembled and consists of a homodimer of hP1. And docked and fixed with _two identical peripheral modules (X ₂ ) bound to DDD2 to form a complex consisting of X ₂ (hP1) X ₂ . One particularly promising area for X ₂ (hP1) X ₂ constructs is the delivery of non-viral vectors through the brain blood barrier for gene therapy of brain disease. For example, a X ₂ (hP1) X ₂ construct whose peripheral module is based on an anti-hTfRFab domain can be used to transport a DNA vector encoding a tyrosine hydroxylase gene through the BBB for the treatment of Parkinson's disease. (Pardridge, NeuroRx®, 2005, 2: 129-138).

Example 5 Generation and Use of DDD2-CH2-CH3-DDD3C (Type b Adapter Module) Polypeptides in which DDD2 and DDD3C are fused to the amino and carboxyl termini of the CH2 and CH3 domains of human IgG1, respectively, are self-associated. To form a structure consisting of two Fc subunits linked via a disulfide bond, and when reduced with a reagent containing a thiol, one contains the substance (X) derivatized with AD2, and the other derivatized with AD3 Further, it is docked and fixed with two peripheral modules containing the prepared substance (Y) to form a complex composed of X (Fc) Y. If the two substances bound to AD are each derived from Fab domains with different specificities, the resulting assembly is an IgG-like bispecific antibody containing a complete Fc domain.

Example 6 Production and Use of DDD2-CH2-CH3-AD2 (Type c Adapter Module) Polypeptides in which DDD3 and AD2 are fused to the amino and carboxyl termini of the CH2 and CH3 domains of human IgG1, respectively, are self-associated. Thus, a structure containing an Fc domain is formed, and docked and fixed with _two identical peripheral modules (X ₂ ) bound to DDD2, to form a complex consisting of X ₂ (Fc) X ₂ .

Example 7 Production and Use of Polypeptides Containing DDD3C-CH2-CH3-AD2 (Type d Adapter Module) A polypeptide in which DDD3C and AD2 are fused to the amino and carboxyl termini of the CH2 and CH3 domains of human IgG1, respectively, The same peripheral module that self-assembles to form a structure composed of two Fc subunits linked via disulfide bonds, reduces one Fc subunit with a thiol-containing reagent, and two are linked to DDD3C (X ₂ ), and the third one is docked and fixed with three peripheral modules, which are substances (Y) bound to AD3, to form a complex consisting of X ₂ (Fc) YX ₂ .

Example 8 Molecular modification of DDD3-CH2-CH3-AD2 and DDD3C-CH2-CH3-AD2 To generate DDD3 and DDD3C sequences, a human RIa cDNA clone (IMAGE clone # 5531156 from Invitrogen) was used as a template 2 Two PCR reactions were performed. In both reactions, the oligonucleotide RI BgIII right was used as the 3 ′ PCR primer. For DDD3 and DDD3C, RI BspHI left and RI-C BspHI left were used as 5 ′ PCR primers, respectively.

Fc domains (CH2 and CH3 domains) were amplified using the pdHL2 vector as a template and Fc domain BgIII left and Fc domain Bam-RcoRI right as primers.

  Each of the amplimers was cloned in the pGemT PCR cloning vector. Using BgIII and EcoRI restriction enzymes, the Fc insert is excised from pGemT and cloned into the same site of the SV3 shuttle vector to generate the intermediate clone Fc-SV3.

  The inserted DDD3 and DDD3C were then excised from pGemT using BspHI and BgIII, ligated with Ncol (end compatible with BspHI) and BgIII digested, and shuttle vector DDD3-Fc-SV3 and DDD3C-Fc-SV3 was generated respectively. Finally, the expression cassette was excised from the SV3 shuttle vector using Xbal and BamHI, and the h679-AD2-pdHL2 vector was ligated with the AD2-pdHL2 vector prepared by digestion with Xbal and BamHI.

  The amino acid sequence of DDD3-Fc-AD2 is shown in FIG. The sequence of DDD3-Fc-AD2 is identical except that the five amino terminal residues of DDD3C-Fc-AD2, namely MSCGG, are replaced by MS.

  Both expression vectors are transfected into sp / EEE cells. Positive clones are screened by ELISA using capture protein-A coated plate and detection HRP-conjugated protein. Purification is performed using protein-A affinity chromatography.

Example 9 Treatment of focal transient cerebral ischemia with a drug comprising a monoclonal antibody to DDD3C-CH2-CH3-AD2 bound to brain-derived neurotrophic factor (BDNF) and human transferrin receptor (hTfR) Within 1 hour of administration, a 10 mg saline solution containing 4 BDNF-DDD2 modules and one anti-hTfRFab-AD3 module stably bound to the DDD3C-Fc-AD2 module is administered intravascularly to the patient TF. Timely treatment reduces the volume of the infarcted part of the whole hemisphere, as shown by MRI, and 48 hours when the symptoms of partial limb paralysis, speech impairment, and confusion are receiving other care and anticoagulant therapy Within a notable improvement.

DDD2 (SEQ ID NO: 1), AD2 (SEQ ID NO: 2), DDD3 (SEQ ID NO: 3), DDD3C (SEQ ID NO: 4), and AD3 (SEQ ID NO: 5) useful for the formation of bioactive assemblies. Examples of such peptide sequences are shown. The composition and use of the sequence to form a bioactive assembly is discussed below. For example, a schematic representation of an X ₂ (Ma) Y ₂ bioactive assembly based on a type a adapter module (Ma) bound to one molecule of AD2 and AD3, respectively, is shown. AD2 and AD3, for example, act as binding sites for DDD2 and DDD3. These dimerization and docking domains may then be bound to various effectors or binding molecules (X and Y). The result is a heterotetramer containing two different homodimers. For example, a schematic diagram of an X (Mb) ₂ Y bioactive assembly based on a type b adapter module (Mb) bound to one molecule of DDD2 and DDD3, respectively, is shown. Addition of an appropriate anchor domain, eg, AD2 and AD3, that bind to two different effectors results in dimerization and formation of X (Mb) ₂ Y assemblies. For example, a schematic diagram of an X ₂ (Mc) ₂ X ₂ bioactive assembly based on a type c adapter module (Mc) bound to one molecule of DDD3 and AD2 respectively is shown. Dimerization of DDD3 sequences bound to different Mc molecules provides, for example, two anchor sites (AD2) for the binding of homodimers with DDD2 bound to the effector, for example. The result is a homotetramer containing 4 effectors X. FIG. 3 shows a schematic of an X ₂ (Md) ₂ YX ₂ bioactive assembly based on a type d adapter module (Md). The difference from the assembly shown in FIG. 4 is that a DDD3C dimerization and docking domain is used so that it can bind to the AD3 anchor domain bound to effector Y. Has DDD3C (SEQ ID NO: 4), first linker (SEQ ID NO: 6), CH2 (SEQ ID NO: 7), CH3 (SEQ ID NO: 8), second linker (SEQ ID NO: 9), and AD2 (SEQ ID NO: 2) Shows an example of the complete amino acid sequence of the DDD3C-CH2-CH3-AD2 construct (see Example 7).

Claims (14)

a) HSP70, alpha ₂ macroglobulin, HSA (human serum albumin), hPl (human protamine 1), heat shock protein is selected from the group consisting of Fc fragments of human protamine and human antibodies, the adapter module;
b) one or more anchor domains (AD) and / or dimerization and docking domains (DDD) bound to the adapter module, where AD is a peptide derived from the anchor domain of AKAP (A-kinase anchor protein) And DDD has a peptide sequence derived from the dimerization and docking domain of human protein kinase A regulatory subunits RIα, RIβ, RIIα or RIIβ; and c) is a protein or peptide, AD or DDD A Dock and Lock (DNL) complex comprising at least one effector coupled to
Wherein the DNL complex comprises at least two DDDs and at least one AD, and the two DDDs form a dimer that binds to AD to form a DNL complex, wherein the DNL complex Is
The adapter module binds to AD2 (SEQ ID NO: 2) and AD3 (SEQ ID NO: 5), and the complex further binds to the first effector and DDD3C (SEQ ID NO: 4) bound to DDD2 (SEQ ID NO: 1). Two DDD2 (SEQ ID NO: 1) form a dimer that binds to AD2 (SEQ ID NO: 2), and two DDD3C (SEQ ID NO: 4) are AD3 (SEQ ID NO: 5). A DNL complex that forms another dimer that binds to
The adapter module binds to DDD2 (SEQ ID NO: 1) and DDD3C (SEQ ID NO: 4), and the complex further binds AD1 (SEQ ID NO: 2) to the first effector and AD3 (SEQ ID NO: 5). Two DDD2 (SEQ ID NO: 1) form a dimer that binds to AD2 (SEQ ID NO: 2), and two DDD3C (SEQ ID NO: 4) are AD3 (SEQ ID NO: 5). A DNL complex that forms another dimer that binds to
The adapter module binds to AD2 (SEQ ID NO: 2) and DDD3 (SEQ ID NO: 3), and the complex further comprises a first effector bound to DDD2 (SEQ ID NO: 1), where two DDD2 (SEQ ID NO: 1) 1) forms a dimer that binds to AD2 (SEQ ID NO: 2) and two DDD3 (SEQ ID NO: 3) form another dimer; and the DNL complex; (SEQ ID NO: 2) and DDD3C (SEQ ID NO: 4), and the complex further comprises a first effector bound to DDD2 (SEQ ID NO: 1) and a second effector bound to AD3 (SEQ ID NO: 5); Here, two DDD2 (SEQ ID NO: 1) form a dimer that binds to AD2 (SEQ ID NO: 2), and two DDD3C (SEQ ID NO: 4) A DNL complex selected from the group consisting of a DNL complex that forms another dimer that binds to 3 (SEQ ID NO: 5).   The DNL complex according to claim 1, wherein the effector is selected from the group consisting of an antibody, an antigen-binding antibody fragment and a cytokine. The DNL complex of claim 2, wherein the antibody fragment is selected from the group consisting of F (ab) ₂ , F (ab ') ₂ , Fab, Fab', Fv, scFv and single domain (DAB) antibody fragments. .   The DNL complex of claim 2, wherein the antibody or antigen-binding antibody fragment has binding affinity for a tumor-associated antigen.   The complex is anti-CD74 antibody × anti-CD20 antibody, anti-CD74 antibody × anti-CD22 antibody, anti-CD22 antibody × anti-CD20 antibody, anti-CD20 antibody × anti-HLA-DR antibody, anti-CD19 antibody × anti-CD20 antibody, anti-CD20 antibody × A first effector and a second effector which are antibodies or antigen-binding antibody fragments selected from the group consisting of anti-CD80 antibody, anti-CD2 antibody × anti-CD25 antibody, anti-CD8 antibody × anti-CD25 antibody, and anti-CD2 antibody × anti-CD147 antibody The DNL complex according to claim 2, comprising: The effectors are LL1 (anti-CD74 antibody), LL2 (anti-CD22 antibody), A20 (anti-CD20 antibody), L243 (anti-HLA class II), CC49 (anti-TAG72), MN-14 (anti-CEA), MN-15 ( anti CEA), L19 (anti-ED-B fibronectin), R1 (anti-IGF-1R), PAM4 (anti MUC1), RS7 (antibody or antigen-binding antibody fragment thereof selected from anti-EGP-1) or Ranaru group The DNL complex according to claim 2.   The DNL complex of claim 2, wherein the antibody or antigen-binding antibody fragment is human, humanized or chimeric.   A pharmaceutical composition comprising the DNL complex according to any one of claims 1 to 7.   The composition is further selected from the group consisting of chemotherapeutic agents, cytokines, chemokines, anti-angiogenic agents, pro-apoptotic agents, pharmaceuticals, prodrugs, toxins, enzymes, boron, radioisotopes, immunomodulators, antibiotics and hormones 9. The composition of claim 8, comprising a therapeutic agent that is treated. Therapeutic agent, abrin, amantadine, amoxicillin, Anfoteshirin B, ampicillin, aplidine, azaribine, anastrozole, azacytidine, aztreonam, azithromycin, bacitracin, bactrim, blanking Reomaishin, bortezomib, bryostatin-1, busulfan, calicheamicin, camptothecin , 10-hydroxy camptothecin, caspofungin, carmustine, cefaclor, cefazolin, cephalosporins, Sefapimu, ceftriaxone, cefotaxime, chlorambucil, chloramphenicol, cisplatin, irinotecan (CPT-11), SN- 38, carboplatin , Cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomy Ngururonide, daunorubicin, dexamethasone, diethylstilbestrol, diphtheria toxin, DNase I, doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX), doxycycline, cyanomorpholino doxorubicin, doxorubi single clonide, epirubising clonide, ethinyl estradiol, estrogen receptor binding agents, etoposide, etoposide glucuronide, etoposide phosphate, erythro cyclin, erythromycin, full § Rene sills protein transferase inhibitor, floxuridine (FUdR), 3 ', 5' -O- dioleoyl FudR (FUdR-dO ), Fludarabine, flutamide, fluorouracil, fluoxymesterone, ganciclovir, gentamicin, Nin, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, isoniazid, itraconazole, kanamycin, ketoconazole, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, minocycline, naftifine, nalidixic acid, neomycin, navelbine, nitrosoureas, nystatin oxacillin, paromomycin, penicillin, pentamidine, piperacillin, phenyl butyric acid, prednisone, Procarbazine, paclitaxel, pentostatin, pokeweed Virus protein, Pseudomonas exotoxin, Pseudomonas aeruginosa endotoxin, raloxifene, rapLR1, ribonuclease, ricin, semustine, rifabutin, vinegar streptomycin, sulfamethoxazole, sulfasalazine, staphylococcal enterotoxin -A, streptozocin, tamoxifen , made Takusanesu, taxol, testosterone propionate, tetracycline, thalidomide, thioguanine, thiotepa, teniposide, topotecan, door Rimetopurimu, sulfamethoxazole, uracil mustard, valacyclovir, vancomycin, Velcade, vinblastine, vinorelbine, vincristine, zanamivir, from Jisuromaishin 10. A composition according to claim 9 selected from the group. Radioactive isotopes are ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ²²³ Ra, ²²⁵ Ac, ³² P, ³³ P, ⁴⁷ Sc, ⁶⁷ Cu, ⁶⁷ Ga, ⁸⁹ Sr, ⁹⁰ Y, ¹¹¹ Ag, ¹²⁵ I, ¹³¹ I, ¹⁴² Pr, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁶⁶ Dy, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, ¹¹¹ In, ¹²⁵ I, ⁶⁷ Ga, ¹⁹¹ Os, ^{193 m} Pt, ^{195 m} Pt and ^{195 m} Pt 10. The composition of claim 9, wherein the composition is selected from the group consisting of:   The toxin is selected from the group consisting of ricin, abrin, ribonuclease, rapLR1, DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas aeruginosa endotoxin. 9. The composition according to 9. The composition further radioisotopes, dyes, contrast agents, Imaging agents include diagnostic agent selected from the group consisting of liposome, and paramagnetic ions, composition of claim 8.   Use of a DNL complex according to claim 1 for the manufacture of a medicament for treating a medical condition.

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