EP2509629A1 - Dock-and-lock (dnl) complexes for delivery of interference rna - Google Patents
Dock-and-lock (dnl) complexes for delivery of interference rnaInfo
- Publication number
- EP2509629A1 EP2509629A1 EP10836672A EP10836672A EP2509629A1 EP 2509629 A1 EP2509629 A1 EP 2509629A1 EP 10836672 A EP10836672 A EP 10836672A EP 10836672 A EP10836672 A EP 10836672A EP 2509629 A1 EP2509629 A1 EP 2509629A1
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- European Patent Office
- Prior art keywords
- seq
- cancer
- sirna
- targeted delivery
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
- C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
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- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C07K2317/00—Immunoglobulins specific features
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- C07K2317/77—Internalization into the cell
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- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3513—Protein; Peptide
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Definitions
- RNAi species such as siRNA
- the targeted delivery complexes comprise an antibody, antibody fragment or other targeting molecule conjugated to a carrier molecule for siRNA, such as a protamine or a dendrimer. More preferably, the targeted delivery complexes are made by the dock-and-lock (DNL) technique.
- the targeted delivery complex may comprise one or more siRNA species for delivery to a targeted cell or tissue. Most preferably, delivery of siRNA is effective to treat a disease, syndrome or disorder, such as cancer, autoimmune disease, immune dysfunction (e.g., graft-versus-host disease or organ transplant rejection), inflammation, infectious disease, cardiovascular disease, endocrine or metabolic disease or neurodegenerative disease.
- the antibody or other targeting molecule binds to a target antigen produced by a diseased cell or tissue or otherwise associated with the disease state.
- the DNL complex may comprise multiple copies of a carrier molecule to efficiently deliver therapeutic siRNA to a target cell.
- the antibody or other targeting molecule may be a rapidly internalizing species to facilitate intracellular uptake of siRNA.
- RNA interference is a naturally occurring regulatory mechanism for control of gene expression in cells (Fire et al., 1998, Nature 391 :806-l 1). RNAi is mediated by the RNA-induced silencing complex (RISC) and is initiated by short double- stranded RNA molecules that interact with the catalytic RISC component argonaute (Rand et al., 2005, Cell 123:621 -29). Types of RNAi molecules include microRNA (miRNA) and small interfering RNA (siRNA). RNAi species can bind with messenger RNA (mRNA) through complementary base-pairing and inhibits gene expression by post- transcriptional gene silencing.
- mRNA microRNA
- siRNA small interfering RNA
- RNAi Upon binding to a complementary mRNA species, RNAi induces cleavage of the mRNA molecule by the argonaute component of RISC.
- miRNA and siRNA differ in the degree of specificity for particular gene targets, with siRNA being relatively specific for a particular target gene and miRNA inhibiting translation of multiple mRNA species.
- RNAi by inhibition of selected gene expression has been attempted for a variety of disease states, such as macular degeneration and respiratory syncytial virus infection (Sah, 2006, Life Sci 79: 1773-80). It has been suggested that siRNA functions in host cell defenses against viral infection and siRNA has been widely examined as an approach to anti-viral therapy (see, e.g., Zhang et al., 2004, Nature Med 1 1 :56-62; Novina et al., 2002, Nature Med 8:681-86; Palliser et al., 2006, Nature 439:89- 94). The use of siRNA for cancer therapy has also been attempted. Fujii et al.
- the present invention concerns methods and compositions for efficient delivery of siRNA species to disease-associated target cells, tissues or pathogens.
- delivery is facilitated by use of targeted delivery complexes, which comprise an antibody, antibody fragment or other targeting molecule conjugated to a carrier molecule for siRNA, such as a protamine, dendrimer or nanoparticle.
- the targeting molecule provides for highly selective or specific delivery of therapeutic siRNA to diseased cells or tissues.
- the targeted delivery complex facilitates intracellular uptake of siRNA into target cells.
- siRNA species to be delivered and the corresponding specificity of the targeting molecule will be determined by the disease state to be treated.
- tens of thousands of siRNA sequences directed to mRNAs associated with a broad variety of disease states are known in the art and may be obtained from public databases, such as siRNAdb database at the Sweden Bioinformatics Centre, the MIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the Broad Institute, and the Probe database at NCBI.
- siRNA species may also be purchased from commercial vendors like Sigma-Aldrich (St Louis, MO), Invitrogen (Carlsbad, CA), Santa Cruz Biotechnology (Santa Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific, Denver, CO), Promega (Madison, WI), Minis Bio (Madison, WI) and Qiagen (Valencia, CA).
- carrier moieties for siRNA have been reported and any such known carrier may be incorporated into a targeted delivery complex for use.
- Non-limiting examples of carriers include protamine (Rossi, 2005, Nat Biotech 23:682-84; Song et al., 2005, Nat Biotech 23:709-17); dendrimers such as PAMAM dendrimers (Pan et al., 2007, Cancer Res. 67:8156-8163); polyethylenimine (Schiffelers et al., 2004, Nucl Acids Res 32:el49); polypropyleneimine (Taratula et al., 2009, J Control Release 140:284-93);
- oligonucleotide or nucleic acid species such as anti-sense oligonucleotides or short DNA genes.
- Various embodiments may concern use of the targeted delivery complexes to treat a disease, including but not limited to non-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T-cell lymphomas and leukemias, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, carcinomas, melanomas, sarcomas, gliomas, and skin cancers.
- carcinomas may be selected from the group consisting of carcinomas of the oral cavity, gastrointestinal tract, colon, stomach, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes.
- the targeted delivery complexes may be used to treat an autoimmune disease, for example acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing
- an autoimmune disease for
- compositions of the present invention also are useful for the treatment of infections, where the antibody component of the targeted delivery complex specifically binds to a disease-causing microorganism.
- a disease-causing microorganism includes pathogenic bacteria, viruses, fungi and diverse parasites, and the antibody can target these microorganisms, their products or antigens associated with their lesions.
- microorganisms include, but are not limited to: Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Haemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, Tetanus toxin, HIV-1, -2, -3, Hepatitis A, B, C, D, Rabies virus, Influenza virus, Cytomegalovirus, Herpes simplex I and II, Human serum parvo-like virus, Papilloma viruses, Polyoma virus, Respiratory syncytial virus, Varicella-Zoster virus,
- a pharmaceutical composition of the present invention may be used to treat a subject having a metabolic disease, such amyloidosis, or a
- a pharmaceutical composition of the present invention may be used to treat a subject having an immune-dysregulatory disorder such as graft-versus-host disease or organ transplant rejection.
- diseases that may be treated using the claimed compositions and methods include cardiovascular diseases, such as fibrin clots, atherosclerosis, myocardial ischemia and infarction.
- cardiovascular diseases such as fibrin clots, atherosclerosis, myocardial ischemia and infarction.
- Antibodies to fibrin e.g.,
- scFv(59D8); T2Gls; MH1) are known and in clinical trials as imaging agents for disclosing said clots and pulmonary emboli, while anti-granulocyte antibodies, such as MN-3, MN-15, anti-NCA95, and anti-CD 15 antibodies, can target myocardial infarcts and myocardial ischemia.
- anti-granulocyte antibodies such as MN-3, MN-15, anti-NCA95, and anti-CD 15 antibodies
- Anti- macrophage, anti-low-density lipoprotein (LDL) and anti-CD74 (e.g., hLLl) antibodies can be used to target atherosclerotic plaques.
- Abciximab anti-glycoprotein Ilb/IIIa
- Anti-CD3 antibodies have been reported to reduce development and progression of atherosclerosis (Steffens et al., 2006, Circulation 1 14: 1977-84).
- Antibodies against oxidized LDL induced a regression of established atherosclerosis in a mouse model (Ginsberg, 2007, J Am Coll Cardiol 52:2319-21).
- Anti-ICAM-1 antibody was shown to reduce ischemic cell damage after cerebral artery occlusion in rats (Zhang et al., 1994, Neurology 44: 1747-51).
- OKT anti-T-cell monoclonal antibodies available from Ortho Pharmaceutical Company which bind to normal T-lymphocytes; the monoclonal antibodies produced by the hybridomas having the ATCC accession numbers HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7, NKP15 and G022 (Becton
- Antibodies or other targeting molecules of use may bind to any disease-associated antigen known in the art.
- the disease state is cancer
- many antigens expressed by or otherwise associated with tumor cells are known in the art, including but not limited to, carbonic anhydrase IX, alpha- fetoprotein, a-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, , CCCL19, CCCL21 , CD1 , CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21 , CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e,
- ELF2-M Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gplOO, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, la, IGF-1R, IFN- ⁇ , IFN- , IFN- ⁇ , IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, S-l-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-
- IGF-1 insulin-like growth factor-1
- RANTES T101 , SAGE, S I 00, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-a, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1 , 17-lA-antigen, complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, an oncogene marker and an oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006, 12:5023-32; Purani et al., J Immunol 2007, 178: 1975-79; Novellino et al. Cancer Immunol Immunother 2005, 54: 187-207).
- Exemplary antibodies that may be utilized include, but are not limited to, hRl (anti-IGF-lR, U.S. Patent Application Serial No. 12/722,645, filed 3/12/10), hPAM4 (anti- mucin, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,251 ,164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No.
- An antibody or antigen-binding fragment of use may be chimeric, humanized or human.
- the use of chimeric antibodies is preferred to the parent murine antibodies because they possess human antibody constant region sequences and therefore do not elicit as strong a human anti-mouse antibody (HAMA) response as murine antibodies.
- HAMA human anti-mouse antibody
- the use of humanized antibodies is even more preferred, in order to further reduce the possibility of inducing a HAMA reaction.
- techniques for humanization of murine antibodies by replacing murine framework and constant region sequences with corresponding human antibody framework and constant region sequences are well known in the art and have been applied to numerous murine anti-cancer antibodies.
- Antibody humanization may also involve the substitution of one or more human framework amino acid residues with the corresponding residues from the parent murine framework region sequences.
- techniques for production of human antibodies are also well known.
- the targeted delivery complex may be produced using the dock-and-lock (DNL) technique (see, e.g., U.S. Patent Nos. 7,550,143;
- DNL dock-and-lock
- the DNL technique utilizes the specific binding interactions occurring between a dimerization and docking domain (DDD moiety) from protein kinase A, and an anchoring domain (AD moiety) from any of a number of known A-kinase anchoring proteins (A APs).
- DDD moieties spontaneously form dimers which then bind to an AD moiety.
- AD and DDD moieties By attaching appropriate effector moieties, such as antibodies or fragments thereof and siRNA carriers, to AD and DDD moieties, the DNL technique allows the specific covalent formation of any desired targeted delivery complex.
- the AD and DDD moieties may be incorporated into fusion proteins conjugated to the effector moieties. Where non-protein effector moieties are utilized, chemical conjugation may be utilized to attach the AD or DDD moiety.
- disease therapy by targeted delivery of siRNA may be enhanced by combination therapy with one or more other therapeutic agents.
- therapeutic agents of use include toxins, immunomodulators (such as cytokines, lymphokines, chemokines, growth factors and tumor necrosis factors), hormones, hormone antagonists, enzymes, oligonucleotides (such as siRNA or RNAi), photoactive therapeutic agents, anti-angiogenic agents and pro-apoptotic agents.
- the therapeutic agents may be delivered by conjugation to the same or different antibodies or other targeting molecules or may be administered in unconjugated form. Other therapeutic agents may be administered before, concurrently with or after the siRNA targeted delivery complex.
- the therapeutic agent is a cytotoxic agent, such as a drug or a toxin.
- the drug is selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, hormone antagonists, endostatin, taxols, camptothecins, SN-38,
- doxorubicins and their analogs, antimetabolites, alkylating agents, antimitotics, anti- angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDAC inhibitors, pro-apoptotic agents, methotrexate, CPT-1 1, calicheamicin and a combination thereof.
- HSP90 heat shock protein
- the therapeutic agent is a toxin selected from the group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin and combinations thereof.
- an immunomodulator selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combinations thereof.
- the therapeutic agent is a radionuclide selected from the group consisting of 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, m Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, l66 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, ,05 Rh, 109 Pd, 143 Pr, I49 Pm, 169 Er, I94 Ir, 198 Au, 199 Au, and 211 Pb.
- a radionuclide selected from the group consisting of 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, m Ag,
- radionuclides that substantially decay with Auger-emitting particles.
- Auger-emitting particles For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-I l l, Sb-1 19, 1-125, Ho-161, Os-189m and Ir- 192.
- Decay energies of useful beta-particle-emitting nuclides are preferably ⁇ 1 ,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV.
- radionuclides that substantially decay with generation of alpha-particles.
- Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
- radioisotopes of use include 1 1 C, I3 N, i5 0, 75 Br, 198 Au, 22 Ac, 126 I, 133 I, 77 Br, 1 13m
- the therapeutic agent is a photoactive therapeutic agent selected from the group consisting of chromogens and dyes.
- the therapeutic agent is an enzyme selected from the group consisting of malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
- malate dehydrogenase staphylococcal nuclease
- delta-V-steroid isomerase yeast alcohol dehydrogenase
- alpha-glycerophosphate dehydrogenase alpha-glycerophosphate dehydrogenase
- triose phosphate isomerase horseradish peroxidas
- Such enzymes may be used, for example, in combination with prodrugs that are administered in relatively non-toxic form and converted at the target site by the enzyme into a cytotoxic agent.
- a drug may be converted into less toxic form by endogenous enzymes in the subject but may be reconverted into a cytotoxic form by the therapeutic enzyme.
- FIG. 1 Synthesis of El-L-thPl DNL complex for siRNA delivery.
- A An anti- Trop-2 hRS7 antibody was produced with an AD2 moiety attached to the C-terminal end of each antibody heavy chain.
- B A DDD2-L-thPl construct was synthesized with a truncated segment of human protamine 1 and a DDD2 moiety attached respectively to the C- and N-terminal ends of a humanized antibody light chain.
- C Incubation of the hRS7- IgG-AD2 and DDD2-L-thPl under mild reducing conditions resulted in the formation of the protamine-conjugated hRS7 DNL complex, designated El-L-thPl .
- FIG. 2 Gel shift assay for DNA binding by El-L-thPl .
- FIG. 3 Internalization of siRNA mediated by El -L-thPl binding.
- FIG. 4 Apoptosis induced by El-L-thPl internalization of siRNA.
- An antibody refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes)
- immunoglobulin molecule e.g., an IgG antibody
- immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule like an antibody fragment.
- an antibody fragment is a portion of an antibody such as F(ab') 2 , Fab', Fab, Fv, sFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody.
- antibody fragment also includes isolated fragments consisting of the variable regions of antibodies, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins").
- a chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody.
- the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.
- a humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences).
- the constant domains of the antibody molecule are derived from those of a human antibody.
- a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.
- a human antibody is, e.g., an antibody obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
- elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous murine heavy chain and light chain loci.
- the transgenic mice can synthesize human antibodies specific for particular antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7: 13 (1994), Lonberg et al, Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
- a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See for example, McCafferty et al, Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors.
- antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle.
- the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B-cell.
- Phage display can be performed in a variety of formats, for review, see e.g. Johnson and Chiswell, Current Opinion in
- a therapeutic agent is a compound, molecule or atom which is administered separately, concurrently or sequentially with an antibody moiety or conjugated to an antibody moiety, i.e., antibody or antibody fragment, or a subfragment, and is useful in the treatment of a disease.
- therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. Therapeutic agents of use are described in more detail below.
- An immunoconjugate is an antibody, antibody fragment or fusion protein conjugated to at least one therapeutic and/or diagnostic agent.
- siRNA species may be attached to the targeted delivery complexes for delivery to a targeted tissue.
- Many siRNA species against a wide variety of targets are known in the art, and any such known siRNA may be utilized in the claimed methods and compositions.
- Known siRNA species of potential use include those specific for IKK-gamma (U.S. Patent 7,022,828); VEGF, Flt-1 and Flk-l/KDR (U.S. Patent 7,148,342); Bcl2 and EGFR (U.S. Patent 7,541 ,453); CDC20 (U.S. Patent 7,550,572); transducin (beta)-like 3 (U.S.
- Patent 7,576,196 KRAS (U.S. Patent 7,576,197); carbonic anhydrase II (U.S. Patent 7,579,457); complement component 3 (U.S. Patent 7,582,746); interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Patent 7,592,443); survivin (U.S. Patent 7,608,7070); superoxide dismutase 1 (U.S. Patent 7,632,938); MET proto-oncogene (U.S. Patent 7,632,939); amyloid beta precursor protein (APP) (U.S. Patent 7,635,771); IGF-IR (U.S. Patent 7,638,621); ICAM1 (U.S.
- APP amyloid beta precursor protein
- Patent 7,642,349 complement factor B (U.S. Patent 7,696,344)
- p53 (7,781 ,575) and apolipoprotein B (7,795,421), the Examples section of each referenced patent incorporated herein by reference.
- siRNA species are available from known commercial sources, such as Sigma-Aldrich (St Louis, MO), Invitrogen (Carlsbad, CA), Santa Cruz Biotechnology (Santa Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific, Lafayette, CO), Promega (Madison, WI), Mirus Bio (Madison, WI) and Qiagen (Valencia, CA), among many others.
- Other publicly available sources of siRNA species include the siRNAdb database at the Swedish Bioinformatics Centre, the MIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the Broad Institute, and the Probe database at NCBI.
- siRNA species there are 30,852 siRNA species in the NCBI Probe database.
- the skilled artisan will realize that for any gene of interest, either a siRNA species has already been designed, or one may readily be designed using publicly available software tools. Any such siRNA species may be delivered using the subject DNL complexes.
- siRNA species known in the art are listed in Table 1. Although siRNA is delivered as a double-stranded molecule, for simplicity only the sense strand sequences are shown in Table 1.
- Sortilin 1 AGGTGGTGTTAACAGCAGAG SEQ ID NO: 1 1
- Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 12
- Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 13
- IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 17
- CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO:26
- CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO:32 [043] The skilled artisan will realize that Table 1 represents a very small sampling of the total number of siRNA species known in the art, and that any such known siRNA may be utilized in the claimed methods and compositions.
- the targeted delivery complex may comprise a monoclonal antibody (MAb) or antigen-binding antibody fragment.
- MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A or Protein-G Sepharose, size- exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al , "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992). After the initial raising of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.
- a chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody.
- Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject.
- CDRs complementarity-determining regions
- a chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the corresponding variable domains of a human antibody.
- the mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences.
- the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40).
- Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005).
- the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
- RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991 , J. Mol. Biol. 222:581-97).
- Human antibodies may also be generated by in vitro activated B-cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, incorporated herein by reference in their entirety. The skilled artisan will realize that these techniques are exemplary and any known method for making and screening human antibodies or antibody fragments may be utilized.
- transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols.
- Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al, Nature 368:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
- a non-limiting example of such a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231 : 11-23, incorporated herein by reference) from Abgenix (Fremont, CA).
- the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
- the XenoMouse® was transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences, along with accessory genes and regulatory sequences.
- the human variable region repertoire may be used to generate antibody producing B-cells, which may be processed into hybridomas by known techniques.
- a XenoMouse® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
- a variety of strains of XenoMouse® are available, each of which is capable of producing a different class of antibody.
- Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
- the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
- the targeted delivery complexes are of use for treatment of malignant disease, cardiovascular disease, infectious disease, inflammatory disease, autoimmune disease, metabolic disease, immune dysfunction disease (e.g., graft versus host disease or organ transplant rejection) or neurological (e.g., neurodegenerative) disease.
- Exemplary target antigens of use for treating such diseases may include carbonic anhydrase IX, CCCL19, CCCL21 , CSAp, CD1 , CDla, CD2, CD3, CD4, CD5, CD8, CD1 1A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7, CXCL12, HIF- la, AFP, PSMA, CEACAM5, CEACAM6, c-met, B7, ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor,
- antibodies of use may target tumor-associated antigens.
- These antigenic markers may be substances produced by a tumor or may be substances which accumulate at a tumor site, on tumor cell surfaces or within tumor cells.
- tumor-associated markers are those disclosed by
- TAAs tumor associated antigens
- Tumor-associated markers have been categorized by Herberman, supra, in a number of categories including oncofetal antigens, placental antigens, oncogenic or tumor virus associated antigens, tissue associated antigens, organ associated antigens, ectopic hormones and normal antigens or variants thereof.
- a sub-unit of a tumor- associated marker is advantageously used to raise antibodies having higher tumor- specificity, e.g., the beta-subunit of human chorionic gonadotropin (HCG) or the gamma region of carcinoembryonic antigen (CEA), which stimulate the production of antibodies having a greatly reduced cross-reactivity to non-tumor substances as disclosed in U.S. Pat. Nos. 4,361,644 and 4,444,744.
- TACI transmembrane activator and CAML-interactor
- B- cell malignancies e.g., lymphoma
- TACI and B-cell maturation antigen BCMA
- APRIL proliferation-inducing ligand
- APRIL stimulates in vitro proliferation of primary B and T-cells and increases spleen weight due to accumulation of B-cells in vivo.
- APRIL also competes with TALL-I (also called BLyS or BAFF) for receptor binding.
- Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B-cells.
- BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity.
- APRIL-TALL-I and BCMA-TACI form a two ligand-two receptor pathway involved in stimulation of B and T- cell function.
- targeted antigens may be selected from the group consisting of CD4, CD5, CD8, CD 14, CD15, CD19, CD20, CD21 , CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, CXCR4, B7, MUC1 , la, Ii, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, an oncogene (e.g., c-met or PLAGL2), an oncogene product, CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).
- any antibody or fragment known in the art that has binding specificity for a target antigen associated with a disease state or condition may be utilized.
- Such known antibodies include, but are not limited to, hRl (anti-IGF-lR, U.S. Patent Application Serial No. 12/772,645, filed 3/12/10) hPAM4 (anti-pancreatic cancer mucin, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,251,164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No.
- hRS7 anti-EGP-1, U.S. Patent No. 7,238,785
- hMN-3 anti- CEACAM6, U.S. Patent No. 7,541 ,440
- Abl24 and Abl25 anti-CXCR4, U.S. Patent No. 7,138,496
- Antibodies of use may be commercially obtained from a wide variety of known sources.
- a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
- a large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions. See, e.g., U.S. Patent Nos. 7,312,318; 7,282,567; 7,151 ,164; 7,074,403; 7,060,802; 7,056,509;
- antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest.
- the antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art.
- Antibody fragments are antigen binding portions of an antibody, such as F(ab') 2 , Fab', F(ab) 2 , Fab, Fv, sFv, scFv and the like. Antibody fragments which recognize specific epitopes can be generated by known techniques. F(ab') 2 fragments, for example, can be produced by pepsin digestion of the antibody molecule. These and other methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331 ,647 and references contained therein. Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 1 19 (1959), Edelman et al, in METHODS IN
- Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246: 1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.
- a single chain Fv molecule comprises a VL domain and a VH domain.
- the VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L).
- L peptide linker
- a scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule.
- VHH Single domain antibodies
- Single domain antibodies may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques.
- the VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs.
- Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (Maass et al., 2007).
- Alpacas may be immunized with known antigens, such as TNF-a, and VHHs can be isolated that bind to and neutralize the target antigen (Maass et al., 2007).
- PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al, 2007).
- An antibody fragment can also be prepared by proteolytic hydrolysis of a full- length antibody or by expression in E. coli or another host of the DNA coding for the fragment.
- An antibody fragment can be obtained by pepsin or papain digestion of full- length antibodies by conventional methods.
- an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an approximate 100 Kd fragment denoted F(ab') 2 .
- This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce an approximate 50 Kd Fab' monovalent fragment.
- an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
- VK variable light chain
- VH variable heavy chain sequences for an antibody of interest
- the V genes of a MAb from a cell that expresses a murine MAb can be cloned by PCR amplification and sequenced.
- the cloned V L and V H genes can be expressed in cell culture as a chimeric Ab as described by Orlandi et al , (Proc. Natl. Acad. Set, USA, 86: 3833 (1989)).
- a humanized MAb can then be designed and constructed as described by Leung et al. (Mol Immunol. , 32: 1413 (1995)).
- cDNA can be prepared from any known hybridoma line or transfected cell line producing a murine MAb by general molecular cloning techniques (Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed (1989)).
- the VK sequence for the MAb may be amplified using the primers VK1BAC and VK1FOR (Orlandi et al , 1989) or the extended primer set described by Leung et al. (BioTechniques, 15: 286 (1993)).
- VH sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandi et al, 1989) or the primers annealing to the constant region of murine IgG described by Leung et al. (Hybridoma, 13:469 (1994)).
- Humanized V genes can be constructed by a combination of long oligonucleotide template syntheses and PCR amplification as described by Leung et al. ⁇ Mol Immunol , 32: 1413 (1995)).
- PCR products for VK can be subcloned into a staging vector, such as a pBR327- based staging vector, VKpBR, that contains an Ig promoter, a signal peptide sequence and convenient restriction sites.
- PCR products for VH can be subcloned into a similar staging vector, such as the pBluescript-based VHpBS.
- Expression cassettes containing the VK and VH sequences together with the promoter and signal peptide sequences can be excised from VKpBR and VHpBS and ligated into appropriate expression vectors, such as pKh and pGlg, respectively (Leung et al., Hybridoma, 13 :469 (1994)).
- the expression vectors can be co-transfected into an appropriate cell and supernatant fluids monitored for production of a chimeric, humanized or human MAb.
- the VK and VH expression cassettes can be excised and subcloned into a single expression vector, such as pdHL2, as described by Gillies et al. (J. Immunol. Methods 125: 191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997)).
- expression vectors may be transfected into host cells that have been pre-adapted for transfection, growth and expression in serum-free medium.
- Exemplary cell lines that may be used include the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Patent Nos. 7,531 ,327; 7,537,930 and 7,608,425; the Examples section of each of which is incorporated herein by reference). These exemplary cell lines are based on the Sp2/0 myeloma cell line, transfected with a mutant Bcl-EEE gene, exposed to methotrexate to amplify transfected gene sequences and pre-adapted to serum- free cell line for protein expression.
- targeted delivery complexes may be produced using the dock-and-lock technology (see, e.g., U.S. Patent Nos. 7,550,143; 7,521 ,056; 7,534,866; 7,527,787 and 7,666,400, the Examples section of each incorporated herein by reference).
- the DNL method exploits specific protein/protein interactions that occur between the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959).
- PKA which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunits
- the structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has a and ⁇ isoforms (Scott, Pharmacol. Ther.
- dimerization domain has been shown to consist of the first 44 amino-terminal residues (Newlon et al, Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et al, J. Biol. Chem. 1990;265;21561)
- AKAP microtubule-associated protein-2
- the amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII dimers ranging from 2 to 90 nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003; 100:4445).
- AKAPs will only bind to dimeric R subunits.
- the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6:216).
- the dimerization domain and AKAP binding domain of human Rlla are both located within the same N-terminal 44 amino acid sequence (Newlon et al, Nat. Struct. Biol. 1999;6:222; Newlon et al, EMBO J. 2001 ;20: 1651), which is termed the DDD herein.
- Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b.
- the dimeric motif of DDD contained in a 2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a 2 and b to form a binary, trimeric complex composed of a 2 b.
- This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et al, Proc. Natl. Acad. Sci. USA.
- DNL constructs of different stoichiometry may be produced and used, including but not limited to dimeric, trimeric, tetrameric, pentameric and hexameric DNL constructs (see, e.g., U.S. Nos. 7,550,143; 7,521 ,056; 7,534,866;
- fusion proteins A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest.
- double-stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
- the AD and/or DDD moiety may be attached to either the N-terminal or C-terminal end of an effector protein or peptide.
- site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity.
- Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
- a moiety such as a siRNA carrier moiety may be covalently attached to an antibody or antibody fragment to form an immunoconjugate.
- Carrier moieties may be attached, for example to reduced SH groups and/or to carbohydrate side chains.
- a carrier moiety can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
- such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2- pyridyldithio)propionate (SPDP). Yu et al, Int. J. Cancer 56: 244 (1994).
- SPDP N-succinyl 3-(2- pyridyldithio)propionate
- MONOCLONAL ANTIBODIES PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in MONOCLONAL ANTIBODIES:
- the carrier moiety can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
- the Fc region may be absent if the antibody component of the immunoconjugate is an antibody fragment.
- a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al, J. Immunol. 154: 5919 (1995); U.S. Patent Nos. 5,443,953 and 6,254,868, the Examples section of which is incorporated herein by reference.
- the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
- click chemistry reaction An alternative method for attaching carrier moieties to a targeting molecule involves use of click chemistry reactions.
- the click chemistry approach was originally conceived as a method to rapidly generate complex substances by joining small subunits together in a modular fashion.
- Various forms of click chemistry reaction are known in the art, such as the Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which is often referred to as the "click reaction.”
- Other alternatives include cycloaddition reactions such as the Diels- Alder, nucleophilic substitution reactions (especially to small strained rings like epoxy and aziridine compounds), carbonyl chemistry formation of urea compounds and reactions involving carbon-carbon double bonds, such as alkynes in thio
- the azide alkyne Huisgen cycloaddition reaction uses a copper catalyst in the presence of a reducing agent to catalyze the reaction of a terminal alkyne group attached to a first molecule.
- a second molecule comprising an azide moiety
- the azide reacts with the activated alkyne to form a 1 ,4-disubstituted 1 ,2,3-triazole.
- the copper catalyzed reaction occurs at room temperature and is sufficiently specific that purification of the reaction product is often not required.
- a copper-free click reaction has been proposed for covalent modification of biomolecules.
- the copper- free reaction uses ring strain in place of the copper catalyst to promote a [3 + 2] azide- alkyne cycloaddition reaction (Id.)
- cyclooctyne is an 8-carbon ring structure comprising an internal alkyne bond.
- the closed ring structure induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole.
- cyclooctyne derivatives may be used for copper-free click reactions (Id. )
- Various embodiments concern methods of treating a cancer in a subject, comprising administering a therapeutically effective amount of a targeted delivery complex, such as an antibody conjugated to a siRNA carrier and loaded with siR A moieties.
- a targeted delivery complex such as an antibody conjugated to a siRNA carrier and loaded with siR A moieties.
- Multimodal therapies may include therapy with other antibodies, such as anti-CD22, anti-CD 19, anti-CD20, anti-CD21, anti-CD74, anti-CD80, anti-CD23, anti-CD45, anti-CD46, anti-MIF, anti- EGP-1 , anti-CEACAM5, anti-CEACAM6, anti-pancreatic cancer mucin, anti-IGF-lR or anti-HLA-DR (including the invariant chain) antibodies in the form of naked antibodies, fusion proteins, or as immunoconjugates.
- antibodies such as anti-CD22, anti-CD 19, anti-CD20, anti-CD21, anti-CD74, anti-CD80, anti-CD23, anti-CD45, anti-CD46, anti-MIF, anti- EGP-1 , anti-CEACAM5, anti-CEACAM6, anti-pancreatic cancer mucin, anti-IGF-lR or anti-HLA-DR (including the invariant chain) antibodies in the form of naked antibodies, fusion proteins, or as immunoconju
- CVB 1.3 g/m 2 cyclophosphamide, 200-400 mg/m 2 etoposide, and 150-200 mg/m 2 carmustine
- CVB 1.5 g/m 2 cyclophosphamide, 200-400 mg/m 2 etoposide, and 150-200 mg/m 2 carmustine
- Other suitable combination chemotherapeutic regimens are well-known to those of skill in the art. See, for example, Freedman et al, "Non-Hodgkin's Lymphomas," in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al.
- first generation chemotherapeutic regimens for treatment of intermediate-grade non-Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone).
- a useful second generation chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin), while a suitable third generation regimen is MACOP-B (methotrexate, doxorubicin,
- cyclophosphamide cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin.
- Additional useful drugs include phenyl butyrate, bendamustine, and bryostatin-1.
- both chemotherapeutic drugs and cytokines are co-administered with a targeted delivery complex.
- the cytokines, chemotherapeutic drugs and targeted delivery complex can be administered in any order, or together.
- Targeted delivery complexes can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the targeted delivery complex is combined in a mixture with a pharmaceutically suitable excipient.
- Sterile phosphate- buffered saline is one example of a pharmaceutically suitable excipient.
- Other suitable excipients are well-known to those in the art. See, for example, Ansel et al. ,
- the targeted delivery complex of the present invention can be formulated for intravenous administration via, for example, bolus injection or continuous infusion.
- the targeted delivery complex is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
- the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs.
- Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added
- compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- the targeted delivery complex may also be administered to a mammal
- the administration may be by continuous infusion or by single or multiple boluses.
- the targeted delivery complex is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
- the dosage of an administered targeted delivery complex for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. It may be desirable to provide the recipient with a dosage of targeted delivery complex that is in the range of from about 1 mg kg to 25 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
- the dosage may be repeated as needed, for example, once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as every other week for several months, or monthly or quarterly for many months, as needed in a maintenance therapy.
- a targeted delivery complex may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages. Or, the targeted delivery complex may be administered twice per week for 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may be administered once or even twice weekly for 4 to 10 weeks.
- the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months. It has been determined, however, that even higher doses, such as 20 mg/kg once weekly or once every 2-3 weeks can be administered by slow i.v. infusion, for repeated dosing cycles.
- the dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
- the subject targeted delivery complexes are of use for therapy of cancer.
- cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies.
- squamous cell cancer e.g., epithelial squamous cell cancer
- Ewing sarcoma e.g., Ewing sarcoma
- Wilms tumor astrocytomas
- lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, as well as head-and-neck cancer.
- cancer includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
- primary malignant cells or tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor
- secondary malignant cells or tumors e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor.
- cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
- Lymphocytic Leukemia Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute
- Lymphocytic Leukemia Adult Acute Myeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related
- Lymphoma Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood
- Lymphoblastic Leukemia Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid
- Osteosarcoma/Malignant Fibrous Histiocytoma of Bone Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Polycythemia vera, Parathyroid Cancer, Penile Cancer,
- Pheochromocytoma Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma
- compositions described and claimed herein may be used to treat malignant or premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
- Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).
- Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia. It is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
- Dysplastic disorders which can be treated include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epi
- Additional pre-neoplastic disorders which can be treated include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
- benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia
- leukoplakia keratoses
- Bowen's disease keratoses
- Farmer's Skin Farmer's Skin
- solar cheilitis solar keratosis
- the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
- Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblasts, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and
- liposarcoma liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
- endotheliosarcoma lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngiom
- a wide variety of therapeutic reagents can be administered concurrently or sequentially with the targeted delivery complex.
- drugs, toxins, and others can be administered concurrently or sequentially with the targeted delivery complex.
- toxins for example, drugs, toxins, and others.
- oligonucleotides immunomodulators, hormones, hormone antagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesis inhibitors, etc.
- the therapeutic agents recited here are those agents that also are useful for administration separately with a targeted delivery complex as described above.
- Therapeutic agents include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti- angiogenic and pro-apoptotic agents, particularly doxorubicin, methotrexate, taxol, CPT- 1 1 , camptothecans, proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinase inhibitors, and others.
- chemotherapeutic drugs such as vinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti- angiogenic and pro-apoptotic agents, particularly doxorubicin, methotrexate, taxol, C
- cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, antimetabolites, pyrimidine analogs, purine analogs, platinum coordination complexes, mTOR inhibitors, tyrosine kinase inhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins, hormones, and the like.
- Suitable chemotherapeutic agents are described in
- conjugates of camptothecins and related compounds may be conjugated to an anti-cancer antibody, for example as disclosed in U.S. Patent No. 7,591 ,994; and USSN 11/388,032, filed March 23, 2006, the Examples section of each of which is incorporated herein by reference.
- a toxin can be of animal, plant or microbial origin.
- a toxin such as Pseudomonas exotoxin, may also be complexed to or form the therapeutic agent portion of an immunoconjugate.
- Other toxins include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
- the term "immunomodulator” includes a cytokine, a lymphokine, a monokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, a transforming growth factor (TGF), TGF-a, TGF- ⁇ , insulin-like growth factor (IGF), erythropoietin,
- thrombopoietin tumor necrosis factor (TNF), TNF- a, TNF- ⁇ , a mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon- a, interferon- ⁇ , interferon- ⁇ , S I factor, IL-1 , IL-lcc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-1 8 IL-21 and IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostat
- the targeted delivery complex may be administered with an immunoconjugate comprising one or more radioactive isotopes useful for treating diseased tissue.
- Particularly useful therapeutic radionuclides include, but are not limited to 1 1 'in, 177 Lu, 2I2 Bi, 213 Bi, 2U At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, ,31 1, 32 P, 33 P, 47 Sc, l u Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, l66 Dy, 166 Ho, 186 Re, , 88 Re, 189 Re, 2l2 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 1 ,)9 Pd, ,43 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, and 211 Pb.
- the therapeutic therapeutic radionuclides include, but are not limited to 1 1 'in, 177 Lu, 2I2 Bi, 213 Bi, 2U At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I
- radionuclide preferably has a decay energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.
- Maximum decay energies of useful beta-particle- emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles.
- beta- particle-emitting nuclides are preferably ⁇ 1 ,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV. Also preferred are radionuclides that substantially decay with generation of alpha-particles.
- Such radionuclides include, but are not limited to: Dy-152, At-21 1, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1 , Ac-225, Fr-221 , At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
- Additional potential therapeutic radioisotopes include U C, 13 N, 15 0, 75 Br, 198 Au, 224 Ac, 126 I, !33 1, 77 Br, 1 13m In, 95 Ru, 97 Ru, l03 Ru, 105 Ru, 107 Hg, 203 Hg, !21m 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 T1, 225 Ac, 76 Br, 169 Yb, and the like.
- the targeted delivery complexes to be delivered to a subject can comprise one or more pharmaceutically suitable excipients, one or more additional ingredients, or some combination of these.
- the targeted delivery complex can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the targeted delivery complex is combined in a mixture with a pharmaceutically suitable excipient.
- Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.
- Other suitable excipients are well-known to those in the art.
- the targeted delivery complex can be formulated for intravenous administration via, for example, bolus injection or continuous infusion.
- Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
- the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- kits containing components suitable for treating or diagnosing diseased tissue in a patient.
- Exemplary kits may contain at least one targeted delivery complex as described herein. If the composition containing components for administration is not formulated for delivery via the alimentary canal, such as by oral delivery, a device capable of delivering the kit components through some other route may be included.
- a device capable of delivering the kit components through some other route may be included.
- a targeted delivery complex may be provided in the form of a pre filled syringe or auto injection pen containing a sterile, liquid formulation or lyophilized preparation of antibody (e.g., Kivitz et al., Clin. Ther. 2006, 28: 1619-29).
- the kit components may be packaged together or separated into two or more containers.
- the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution.
- a kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents.
- Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like.
- Kit components may be packaged and maintained sterilely within the containers. Another component that can be included is instructions for use of the kit.
- the DNL technique can be used to make dimers, trimers, tetramers, hexamers, etc. comprising virtually any antibody, antibody fragment, siRNA carrier or other effector moieties.
- the antibodies and carrier moieties of the subject targeted delivery complex may be produced as fusion proteins comprising either a dimerization and docking domain (DDD) or anchoring domain (AD) sequence.
- DDD and AD moieties may be joined to targeting molecules and siRNA carriers as fusion proteins, the skilled artisan will realize that other methods of conjugation exist, particularly for non-protein siRNA carriers, such as chemical cross- linking, click chemistry reaction, etc.
- the technique is not limiting and any protein or peptide of use may be produced as an AD or DDD fusion protein for incorporation into a DNL construct.
- the AD and DDD conjugates may comprise any molecule that may be cross-linked to an AD or DDD sequence using any cross-linking technique known in the art.
- a dendrimer or other polymeric moiety such as polyethyleneimine or polyethylene glycol (PEG), may be incorporated into a DNL construct, as described in further detail below.
- AD or DDD sequences may be utilized. Exemplary DDD and AD sequences are provided below.
- DDD1 SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:33)
- DDD2 CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:34)
- AD2 CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 36) [0113]
- DDDl and DDD2 comprise the DDD sequence of the human Rlla form of protein kinase A.
- the DDD and AD moieties may be based on the DDD sequence of the human RIa form of protein kinase A and a corresponding AKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.
- the plasmid vector pdHL2 has been used to produce a number of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125: 191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.
- the di-cistronic mammalian expression vector directs the synthesis of the heavy and light chains of IgG.
- the vector sequences are mostly identical for many different IgG-pdHL2 constructs, with the only differences existing in the variable domain (VH and VL) sequences. Using molecular biology tools known to those skilled in the art, these IgG expression vectors can be converted into Fab- DDD or Fab-AD expression vectors.
- Fab-DDD expression vectors To generate Fab-DDD expression vectors, the coding sequences for the hinge, CH2 and CH3 domains of the heavy chain are replaced with a sequence encoding the first 4 residues of the hinge, a 14 residue Gly-Ser linker and the first 44 residues of human RIIoc (referred to as DDDl).
- AD1 AKAP-TS
- the CHI domain was amplified by PCR using the pdHL2 plasmid vector as a template.
- the left PCR primer consisted of the upstream (5') end of the CHI domain and a SacII restriction endonuclease site, which is 5 1 of the CHI coding sequence.
- the right primer consisted of the sequence coding for the first 4 residues of the hinge (PKSC) followed by four glycines and a serine, with the final two codons (GS) comprising a Bam HI restriction site.
- the 410 bp PCR amplimer was cloned into the PGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened for inserts in the T7 (5') orientation.
- a duplex oligonucleotide was synthesized to code for the amino acid sequence of DDDl preceded by 1 1 residues of the linker peptide, with the first two codons comprising a BamHI restriction site. A stop codon and an Eagl restriction site are appended to the 3'end.
- the encoded polypeptide sequence is shown below.
- oligonucleotides designated RIIA1-44 top and RIIA1-44 bottom, which overlap by 30 base pairs on their 3' ends, were synthesized and combined to comprise the central 154 base pairs of the 174 bp DDDl sequence.
- the oligonucleotides were annealed and subjected to a primer extension reaction with Taq polymerase. Following primer extension, the duplex was amplified by PCR. The amplimer was cloned into PGEMT® and screened for inserts in the T7 (5') orientation.
- a duplex oligonucleotide was synthesized to code for the amino acid sequence of AD1 preceded by 1 1 residues of the linker peptide with the first two codons comprising a BamHI restriction site. A stop codon and an Eagl restriction site are appended to the 3'end. The encoded polypeptide sequence is shown below.
- AKAP-IS Top Two complimentary overlapping oligonucleotides encoding the above peptide sequence, designated AKAP-IS Top and AKA.P-IS Bottom, were synthesized and annealed. The duplex was amplified by PCR. The amplimer was cloned into the above peptide sequence.
- a 190 bp fragment encoding the DDD1 sequence was excised from PGEMT® with BamHI and Notl restriction enzymes and then ligated into the same sites in CH1- PGEMT® to generate the shuttle vector CHl -DDDl -PGEMT®.
- a 1 10 bp fragment containing the ADl sequence was excised from PGEMT® with BamHI and Notl and then ligated into the same sites in CHI -PGEMT® to generate the shuttle vector CHI -ADl -PGEMT®.
- CHl -DDDl or CHI -ADl can be incorporated into any IgG construct in the pdHL2 vector.
- the entire heavy chain constant domain is replaced with one of the above constructs by removing the SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment of CHl-DDDl or CHI -ADl , which is excised from the respective pGemT shuttle vector.
- h679-Fd-ADl-pdHL2 is an expression vector for production of h679 Fab with ADl coupled to the carboxyl terminal end of the CHI domain of the Fd via a flexible Gly/Ser peptide spacer composed of 14 amino acid residues.
- a pdHL2-based vector containing the variable domains of h679 was converted to h679-Fd- AD 1 -pdHL2 by replacement of the SacII/EagI fragment with the CHI -ADl fragment, which was excised from the CH1-AD1-SV3 shuttle vector with SacII and Eagl.
- C-DDD1 -Fd-hMN- 14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein C-DDDl-Fab-hMN-14, in which DDD1 is linked to hMN- 14 Fab at the carboxyl terminus of CHI via a flexible peptide spacer.
- the plasmid vector hMN-14(I)-pdHL2 which has been used to produce hMN-14 IgG, was converted to C-DDD1 -Fd-hMN- 14-pdHL2 by digestion with SacII and Eagl restriction endonucleases to remove the CH1 -CH3 domains and insertion of the CHl- DDDl fragment, which was excised from the CH1-DDD1 -SV3 shuttle vector with SacII and Eagl.
- AD- and DDD-fusion proteins comprising a Fab fragment of any of such antibodies may be combined, in an approximate ratio of two DDD-fusion proteins per one AD-fusion protein, to generate a trimeric DNL construct comprising two Fab fragments of a first antibody and one Fab fragment of a second antibody.
- N-DDDl -Fd-hMN-14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein N-DDDl-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the amino terminus of VH via a flexible peptide spacer.
- the expression vector was engineered as follows. The DDD1 domain was amplified by PCR.
- the 170 bp PCR amplimer was cloned into the pGemT vector and clones were screened for inserts in the T7 (5') orientation.
- the 194 bp insert was excised from the pGemT vector with Ncol and Sail restriction enzymes and cloned into the SV3 shuttle vector, which was prepared by digestion with those same enzymes, to generate the intermediate vector DDD1-SV3.
- the hMN-14 Fd sequence was amplified by PCR. As a result of the PCR, a BamHI restriction site and the coding sequence for part of the linker were appended to the 5' end of the amplimer. A stop codon and Eagl restriction site was appended to the 3' end. The 1043 bp amplimer was cloned into pGemT. The hMN-14-Fd insert was excised from pGemT with BamHI and Eagl restriction enzymes and then ligated with DDD1-SV3 vector, which was prepared by digestion with those same enzymes, to generate the construct N-DDD 1 -hMN- 14Fd-S V3.
- the N-DDD 1 -hMN- 14 Fd sequence was excised with Xhol and Eagl restriction enzymes and the 1.28 kb insert fragment was ligated with a vector fragment that was prepared by digestion of C-hMN- 14-pdHL2 with those same enzymes.
- the final expression vector was N-DDDl-Fd-hMN-14-pDHL2.
- the N-linked Fab fragment exhibited similar DNL complex formation and antigen binding characteristics as the C- linked Fab fragment (not shown).
- C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of C-DDD2- Fab-hMN-14, which possesses a dimerization and docking domain sequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker.
- the fusion protein secreted is composed of two identical copies of hMN-14 Fab held together by non-covalent interaction of the DDD2 domains.
- the expression vector was engineered as follows. Two overlapping,
- oligonucleotides which comprise the coding sequence for part of the linker peptide and residues 1-13 of DDD2, were made synthetically.
- the oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Pstl, respectively.
- the duplex DNA was ligated with the shuttle vector CH1-DDD1 -PGEMT®, which was prepared by digestion with BamHI and Pstl, to generate the shuttle vector CH1- DDD2-PGEMT®.
- a 507 bp fragment was excised from CH 1 -DDD2-PGEMT® with SacII and Eagl and ligated with the IgG expression vector hMN-14(I)-pdHL2, which was prepared by digestion with SacII and Eagl.
- the final expression construct was designated C-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized to generated DDD2- fusion proteins of the Fab fragments of a number of different humanized antibodies.
- h679-Fab-AD2 was designed to pair as B to C-DDD2-Fab-hMN- 14 as A.
- h679- Fd-AD2-pdHL2 is an expression vector for the production of h679-Fab-AD2, which possesses an anchoring domain sequence of AD2 appended to the carboxyl terminal end of the CHI domain via a 14 amino acid residue Gly/Ser peptide linker.
- AD2 has one cysteine residue preceding and another one following the anchor domain sequence of AD1.
- the expression vector was engineered as follows. Two overlapping,
- AD2 Top and AD2 Bottom which comprise the coding sequence for AD2 and part of the linker sequence, were made synthetically.
- the oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Spel, respectively.
- duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, which was prepared by digestion with BamHI and Spel, to generate the shuttle vector CH1-AD2- PGEMT®.
- a 429 base pair fragment containing CHI and AD2 coding sequences was excised from the shuttle vector with SacII and Eagl restriction enzymes and ligated into h679-pdHL2 vector that prepared by digestion with those same enzymes.
- the final expression vector is h679-Fd-AD2-pdHL2.
- TFl DNL construct
- N-DDD2-Fab-hMN- 14 Protein L-purified
- h679-Fab-AD2 IMP-291- purified
- SE-HPLC did not show any evidence of a 2 b formation (not shown). Instead there were peaks representing a 4 (7.97 min; 200 kDa), a 2 (8.91 min; 100 kDa) and B (10.01 min; 50 kDa).
- TFl is a highly stable complex.
- HSG HSG
- N-DDD2-Fab-hMN-14 and 679-Fab-AD2 were mixed and reduced with 5 mM TCEP for 1 hour at room temperature.
- the solution was brought to 0.75 M ammonium sulfate and then loaded onto a Butyl FF HIC column.
- the column was washed with 0.75 M ammonium sulfate, 5 mM EDTA, PBS to remove TCEP.
- the reduced proteins were eluted from the HIC column with PBS and brought to 10% DMSO. Following incubation at room temperature overnight, highly purified TFl was isolated by IMP-291 affinity chromatography (not shown). No additional purification steps, such as gel filtration, were required.
- a trimeric DNL construct designated TF2 was obtained by reacting C-DDD2-Fab- hMN-14 with h679-Fab-AD2.
- a pilot batch of TF2 was generated with >90% yield as follows.
- Protein L-purified C-DDD2-Fab-hMN- 14 200 mg was mixed with h679-Fab- AD2 (60 mg) at a 1.4: 1 molar ratio.
- the total protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.
- Subsequent steps involved TCEP reduction, HIC chromatography, DMSO oxidation, and IMP 291 affinity chromatography. Before the addition of TCEP, SE-HPLC did not show any evidence of a 2 b formation.
- TF2 was purified to near homogeneity by IMP 291 affinity chromatography (not shown).
- IMP 291 is a synthetic peptide containing the HSG hapten to which the 679 Fab binds (Rossi et al, 2005, Clin Cancer Res 1 1 :7122s- 29s).
- SE-HPLC analysis of the IMP 291 unbound fraction demonstrated the removal of a 4 , a 2 and free kappa chains from the product (not shown).
- TF2 The functionality of TF2 was determined by BIACORE® assay.
- TF2, C-DDD1 - hMN-14+h679-ADl (used as a control sample of noncovalent a 2 b complex), or C-DDD2- hMN-14+h679-AD2 (used as a control sample of unreduced a 2 and b components) were diluted to 1 ⁇ g/ml (total protein) and passed over a sensorchip immobilized with HSG.
- the response for TF2 was approximately two-fold that of the two control samples, indicating that only the h679-Fab-AD component in the control samples would bind to and remain on the sensorchip.
- TF10 DNL construct comprising two copies of a C-DDD2-Fab-hPAM4 and one copy of C-AD2-Fab-679.
- the cancer-targeting antibody component in TF10 was derived from hPAM4, a humanized anti-pancreatic cancer mucin MAb that has been studied in detail as a radiolabeled MAb (e.g., Gold et al., Clin. Cancer Res. 13 : 7380-7387, 2007).
- the hapten-binding component was derived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG) MAb.
- the TF10 bispecific ([hPAM4] 2 x h679) antibody was produced using the method disclosed for production of the (anti CEA) 2 x anti HSG bsAb TF2, as described above.
- the TF10 construct bears two humanized PAM4 Fabs and one humanized 679 Fab.
- the two fusion proteins (hPAM4-DDD and h679-AD2) were expressed independently in stably transfected myeloma cells.
- the tissue culture supernatant fluids were combined, resulting in a two-fold molar excess of hPAM4-DDD.
- the reaction mixture was incubated at room temperature for 24 hours under mild reducing conditions using 1 mM reduced glutathione. Following reduction, the DNL reaction was completed by mild oxidation using 2 mM oxidized glutathione.
- TF10 was isolated by affinity chromatography using IMP 291-affigel resin, which binds with high specificity to the h679 Fab.
- DNL techniques may be used to produce complexes comprising any combination of antibodies, immunoconjugates, siRNA carrier moieties or other effector moieties that may be attached to an AD or DDD moiety.
- the IgG and Fab fusion proteins shown in Table 2 were constructed and incorporated into DNL constructs.
- the fusion proteins retained the antigen-binding characteristics of the parent antibodies and the DNL constructs exhibited the antigen-binding activities of the incorporated antibodies or antibody fragments.
- the AD and DDD sequences incorporated into the DNL construct comprise the amino acid sequences of ADl , AD2, AD3, DDDl , DDD2, DDD3 or DDD3C as discussed above.
- sequence variants of AD and/or DDD moieties may be utilized in construction of the DNL complexes.
- the Rlla DDD sequence is the basis of DDD 1 and DDD2 disclosed above.
- the four human PKA DDD sequences are shown below.
- the DDD sequence represents residues 1-44 of Rlla, 1-44 of RIIp, 12-61 of RIa and 13-66 of Rip. (Note that the sequence of DDD1 is modified slightly from the human PKA Rlla DDD moiety.)
- Alto et al. (2003) performed a bioinformatic analysis of the AD sequence of various AKAP proteins to design an RII selective AD sequence called AKAP-IS (SEQ ID NO:35), with a binding constant for DDD of 0.4 nM.
- the AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD are underlined in SEQ ID NO:35.
- sequence variants of the AD sequence one would desirably avoid changing any of the underlined residues, while conservative amino acid substitutions might be made for residues that are less critical for DDD binding.
- Gold (2006) utilized crystallography and peptide screening to develop a
- SuperAKAP-IS sequence (SEQ ID NO:46), exhibiting a five order of magnitude higher selectivity for the RII isoform of PKA compared with the RI isoform.
- Underlined residues indicate the positions of amino acid substitutions, relative to the AKAP-IS sequence, which increased binding to the DDD moiety of Rlla.
- the N-terminal Q residue is numbered as residue number 4 and the C-terminal A residue is residue number 20.
- Residues where substitutions could be made to affect the affinity for Rlla were residues 8, 1 1 , 15, 16, 18, 19 and 20 (Gold et al., 2006).
- the SuperAKAP-IS sequence may be substituted for the AKAP- IS AD moiety sequence to prepare DNL constructs.
- Other alternative sequences that might be substituted for the AKAP-IS AD sequence are shown in SEQ ID NO:47-49. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:46, the AD moiety may also include the additional N-terminal residues cysteine and glycine and C-terminal residues glycine and cysteine.
- Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins, shown below.
- LAWKIAKMIVSDVMQQ (SEQ ID NO:59)
- AKAP7 -wt-pep PEDAELVRLSKRLVENAVLKAVQQY SEQ ID NO:66
- AKAP7 ⁇ S-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY SEQ ID NO:67
- AKAP7 ⁇ 5-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY SEQ ID NO:68
- AKAP7 -P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO:69)
- AKAP7 ⁇ -PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 70)
- AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 76)
- AKAP 1 1 -pep VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 77)
- AKAP 12 NGILELETKS SKLVQNIIQTAVDQF (SEQ ID NO:78)
- Carr et al. examined the degree of sequence homology between different AKAP-binding DDD sequences from human and non-human proteins and identified residues in the DDD sequences that appeared to be the most highly conserved among different DDD moieties. These are indicated below by underlining with reference to the human PKA RIIcc DDD sequence of SEQ ID NO:33. Residues that were particularly conserved are further indicated by italics. The residues overlap with, but are not identical to those suggested by Kinderman et al. (2006) to be important for binding to AKAP proteins.
- Cationic polymers such as polylysine, polyethylenimine, or polyamidoamine (PAMAM)-based dendrimers, form complexes with nucleic acids.
- PAMAM polyamidoamine
- One approach to improve selectivity and potency of a dendrimeric nanoparticle may be achieved by conjugation with an antibody that internalizes upon binding to target cells.
- E1-G5/2 We synthesized and characterized a novel immunoconjugate, designated E1-G5/2, which was made by the DNL method to comprise half of a generation 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to a stabilized dimer of Fab derived from hRS7, a humanized antibody that is rapidly internalized upon binding to the Trop-2 antigen expressed on various solid cancers.
- G5/2 generation 5
- Fab derived from hRS7 a humanized antibody that is rapidly internalized upon binding to the Trop-2 antigen expressed on various solid cancers.
- E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2 and hRS7-Fab-DDD2, under mild redox conditions, followed by purification on a Protein L column.
- AD2-G5/2 we derivatized the AD2 peptide with a maleimide group to react with the single thiol generated from reducing a G5 PAMAM with a cystamine core and used reversed-phase HPLC to isolate AD2-G5/2.
- hRS7-Fab-DDD2 as a fusion protein in myeloma cells, as described in the Examples above.
- E1-G5/2 The molecular size, purity and composition of E1-G5/2 were analyzed by size- exclusion HPLC, SDS-PAGE, and Western blotting. The biological functions of E1-G5/2 were assessed by binding to an anti-idiotype antibody against hRS7, a gel retardation assay, and a DNase protection assay.
- E1 -G5/2 was shown by size-exclusion HPLC to consist of a major peak (>90%) flanked by several minor peaks.
- the three constituents of E1-G5/2 (Fd-DDD2, the light chain, and AD2-G5/2) were detected by reducing SDS-PAGE and confirmed by Western blotting.
- Anti-idiotype binding analysis revealed E1-G5/2 contained a population of antibody-dendrimer conjugates of different size, all of which were capable of recognizing the anti-idiotype antibody, thus suggesting structural variability in the size of the purchased G5 dendrimer.
- the DNL technique can be used to build dendrimer-based nanoparticles that are targetable with antibodies.
- Such agents have improved properties as carriers of drugs, plasmids or siRNAs for applications in vitro and in vivo.
- the peptide IMP 498 up to and including the PEG moiety was synthesized on a Protein Technologies PS3 peptide synthesizer by the Fmoc method on Sieber Amide resin (0.1 mmol scale).
- the maleimide was added manually by mixing the ⁇ - maleimidopropionic acid NHS ester with diisopropylethylamine and DMF with the resin for 4 hr.
- the peptide was cleaved from the resin with 15 mL TFA, 0.5 mL H 2 0, 0.5 mL triisopropylsilane, and 0.5 mL thioanisole for 3 hr at room temperature.
- the peptide was purified by reverse phase HPLC using H 2 0/CH 3 CN TFA buffers to obtain about 90 mg of purified product after lyophilization.
- RNA interference has been shown to down-regulate the expression of various proteins such as HER2, VEGF, Raf-1 , bcl-2, EGFR and numerous others in preclinical studies. Despite the potential of RNA/ to silence specific genes, the full therapeutic potential of RNA/ ' remains to be realized due to the lack of an effective delivery system to target cells in vivo.
- a DDD2-L-thPl module comprising truncated human protamine (thPl, residues 8 to 29 of human protamine 1) was produced, in which the sequences of DDD2 and thPl were fused respectively to the N- and C- terminal ends of a humanized antibody light chain (FIG. 1).
- the sequence of the truncated hPl (thPl) is shown below.
- the DNL technique was employed to generate El -L-thPl (FIG. 1).
- the hRS7 IgG-AD module (FIG. 1A), constructed as described in the Examples above, was expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography.
- the DDD2-L-thP 1 module (FIG. IB) was expressed as a fusion protein in myeloma cells and was purified by Protein L affinity chromatography.
- the CH3-AD2-IgG module possesses two AD2 peptides and each can bind to a DDD2 dimer, with each DDD2 monomer attached to a protamine moiety, the resulting El-L-thPl conjugate comprises four protamine groups (FIG. 1C).
- El-L-thpl was formed in nearly quantitative yield from the constituent modules and was purified to near homogeneity (not shown) with Protein A.
- DDD2-L-thPl was purified using Protein L affinity chromatography and assessed by size exclusion HPLC analysis and SDS-PAGE under reducing and nonreducing conditions (data not shown). A major peak was observed at 9.6 min (not shown). SDS- PAGE showed a major band between 30 and 40 kDa in reducing gel and a major band about 60 kDa (indicating a dimeric form of DDD2-L-thPl) in nonreducing gel (not shown). The results of Western blotting confirmed the presence of monomeric DDD2-L- tPl and dimeric DDD2-L-tPl on probing with anti-DDD antibodies (not shown).
- El-L-thPl was prepared by reducing SDS-PAGE (not shown), which showed the presence of all three constituents (AD2-appended heavy chain, DDD2-L-htPl, and light chain).
- DDD2-L-thPl (not shown) and El -L-thPl (FIG. 2) to bind DNA was evaluated by gel shift assay.
- DDD2-L-thPl retarded the mobility of 500 ng of a linear form of 3-kb DNA fragment in 1% agarose at a molar ratio of 6 or higher (not shown).
- FIG. 2 shows that El -L-thPl retarded the mobility of 250 ng of a linear 200-bp DNA duplex in 2% agarose at a molar ratio of 4 or higher (FIG. 2, lanes d-g), whereas no such effect was observed for hRS7-IgG-AD2 alone (FIG. 2, lane a).
- FIG. 3, lower left El-L-thPl was able to effectively promote internalization of bound FITC-conjugated siRNA (FIG. 3, lower right).
- El-L-thPl (10 ⁇ g) was mixed with FITC- siRNA (300 nM) and allowed to form El-L-thPl -siRNA complexes which were then added to Trop-2-expressing Calu-3 cells. After incubation for 4 h at 37°C the cells were checked for internalization of siRNA by fluorescence microscopy (FIG. 3, lower right).
- El-L-thPl The ability of El-L-thPl to induce apoptosis by internalization of siRNA was examined (FIG. 4).
- El-L-thPl (10 ⁇ g) was mixed with varying amounts of siRNA (AllStars Cell Death siRNA, Qiagen, Valencia, CA).
- the El -L-thPl -siRNA complex was added to ME-180 cells. After 72 h of incubation, cells were trypsinized and annexin V staining was performed to evaluate apoptosis (FIG. 4).
- the DNL technology provides a modular approach to efficiently tether multiple protamine molecules to the anti- Trop-2 hRS7 antibody resulting in the novel molecule El -L-thPl .
- SDS-PAGE demonstrated the homogeneity and purity of El-L-thPl .
- DNase protection and gel shift assays showed the DNA binding activity of El -L-thPl .
- El-L-thPl internalized in the cells like the parental hRS7 antibody and was able to effectively internalize siRNA molecules into Trop-2-expressing cells, such as ME-180 and Calu-3.
- the DNL technique is not limited to any specific antibody or siRNA species. Rather, the same methods and compositions demonstrated herein can be used to make targeted delivery complexes comprising any antibody, any siRNA carrier and any siRNA species.
- the use of a bivalent IgG in targeted delivery complexes would result in prolonged circulating half-life and higher binding avidity to target cells, resulting in increased uptake and improved efficacy.
- This approach to siRNA mediated cell death is particularly appropriate for diseases like pancreatic cancer, where present therapies are ineffective.
- Pancreatic cancers are known to express CEACAM6 and CD74, both of which are associated with the invasiveness of pancreatic cancer.
- siRNAs for CD74 sc-35023, Santa Cruz Biotechnology, Santa Cruz, CA
- CEACAM6 sense strand 5'-CCGGACAGUUCCAUGUAUAdTdT-3' (SEQ ID NO:82)
- Sense and antisense siRNAs are dissolved in 30 mM HEPES buffer to a final concentration of 20 ⁇ , heated to 90°C for 1 min and incubated at 37°C for 60 min to form duplex siRNA.
- the duplex siRNA is mixed with El-L-thPl and incubated with BxPC-3 (CEACAM6-siRNA) and Capan2 (CD74-siRNA) cells.
- the changes in the levels of mRNA for the corresponding proteins are determined by real time quantitative PCR analysis.
- the levels CD74 and CEACAM6 proteins are determined by Western blot analysis and immunohistochemistry.
- Controls include nonspecific siRNA and the non-targeting DNL complex 20-L-thPl , which contains a humanized anti-CD20 antibody (hA20).
- Two xenograft models are established in female athymic nu/nu mice (5 weeks of age, weighing 18-20 g).
- the subcutaneous model has BxPC-3 (ATCC No. CRL-1687) and Capan2 (ATCC No. HTB-80) implanted in opposite flanks of each animal with treatment initiated once tumors reach 50 mm 3 .
- the orthotopic model bears only BxPC-3 cells and treatment is started 2 weeks after implantation.
- the efficacy of El -L-thPl to deliver a mixture of CEACAM6- and CD74-siRNAs is assessed and compared to that of El-L-thPl to deliver CEACAM6-, CD74-, or control siRNA individually. Additional controls are saline and the use of 20-L-fhPl instead of El-L-thPl to deliver the specific and control siRNAs.
- the dosage, schedule, and administration are 150 ⁇ 3 ⁇ 4 based on siRNA, twice weekly for 6 weeks, and via tail vein injection (Table 4). Cells are expanded in tissue culture, harvested with Trypsin/EDTA, and re-suspended with matrigel (1 : 1) to deliver 5xl0 6 cells in 300 ⁇ .
- the orthotopic model is set up as follows. Briefly, nude mice are anesthetized and a left lateral abdominal incision is made. The spleen and attached pancreas are exteriorized with forceps. Then 50 of a BxPC-3 cell suspension (2xl0 6 cells) is injected into the pancreas. The spleen and pancreas are placed back into the abdominal cavity and the incision closed. Therapy begins two weeks after implantation. Mice are treated systemically with CEACAM6- or control siRNA bound to El-L-thPl or 20-L-thPl with the same dosing schedule and route as the subcutaneous model. Animals are monitored daily and weighed weekly.
- CD20 antibody is ineffective to induce siRNA uptake or cancer cell death.
Abstract
Description
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US12/644,146 US7981398B2 (en) | 2005-04-06 | 2009-12-22 | PEGylation by the dock and lock (DNL) technique |
US30268210P | 2010-02-09 | 2010-02-09 | |
US12/731,781 US8003111B2 (en) | 2005-04-06 | 2010-03-25 | Dimeric alpha interferon pegylated site-specifically shows enhanced and prolonged efficacy in vivo |
US12/752,649 US8034352B2 (en) | 2005-04-06 | 2010-04-01 | Tetrameric cytokines with improved biological activity |
US12/754,740 US8562988B2 (en) | 2005-10-19 | 2010-04-06 | Strategies for improved cancer vaccines |
US12/869,823 US20110020273A1 (en) | 2005-04-06 | 2010-08-27 | Bispecific Immunocytokine Dock-and-Lock (DNL) Complexes and Therapeutic Use Thereof |
US12/871,345 US8551480B2 (en) | 2004-02-13 | 2010-08-30 | Compositions and methods of use of immunotoxins comprising ranpirnase (Rap) show potent cytotoxic activity |
US12/915,515 US20110064754A1 (en) | 2005-03-03 | 2010-10-29 | Immunoconjugates Comprising Poxvirus-Derived Peptides and Antibodies Against Antigen-Presenting Cells for Subunit-Based Poxvirus Vaccines |
US41459210P | 2010-11-17 | 2010-11-17 | |
US12/949,536 US8211440B2 (en) | 2005-10-19 | 2010-11-18 | Multivalent immunoglobulin-based bioactive assemblies |
PCT/US2010/059660 WO2011072114A1 (en) | 2009-12-09 | 2010-12-09 | Dock-and-lock (dnl) complexes for delivery of interference rna |
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