Compositions and Methods of Use of Immunotoxins Comprising Ranpirnase (Rap) Show Potent Cytotoxic Activity

Abstract

The present invention concerns methods and compositions for forming immunotoxin complexes having a high efficacy and low systemic toxicity. In preferred embodiments, the toxin moiety is a ranpirnase (Rap), such as Rap(Q). In more preferred embodiments, the immunotoxin is made using dock-and-lock (DNL) technology. The immunotoxin exhibits improved pharmacokinetics, with a longer serum half-life and significantly greater efficacy compared to toxin alone, antibody alone, unconjugated toxin plus antibody or even other types of toxin-antibody constructs. In a most preferred embodiment the construct comprises an anti-Trop-2 antibody conjugated to Rap, although other combinations of antibodies, antibody fragments and toxins may be used to form the subject immunotoxins. The immunotoxins are of use to treat a variety of diseases, such as cancer, autoimmune disease or immune dysfunction.

What is claimed is: 
     
         1 . A method of treating cancer, comprising:
 a) obtaining a complex comprising (i) a first fusion protein comprising a toxin attached to a DDD (dimerization and docking domain) moiety from human protein kinase A (PKA) regulatory subunit RIα, RIβ, RIIα or RIIβ, wherein the toxin is selected from the group consisting of ranpirnase (Rap), saporin, diphtheria toxin, gelonin,  Pseudomonas  exotoxin; and (ii) a second fusion protein comprising an antibody or antigen binding antibody fragment attached to an AD (anchoring domain) moiety from an AKAP protein, wherein the antibody or antibody fragment binds to a human tumor-associated antigen (TAA); and   b) administering the complex to a human subject with cancer;   
       wherein two copies of the DDD moiety form a dimer that binds to the AD moiety to form the complex. 
     
     
         2 . The method of  claim 1 , wherein the TAA is selected from the group consisting of carbonic anhydrase IX, CCL19, CCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, 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, AFP, PSMA, CEACAM-5, CEACAM-6, B7, ED-B, fibronectin, Factor H, Flt-1, Flt-3, folate receptor, GRO-β, HMGB-1, hypoxia inducible factor (HIF), insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, 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, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, NCA-95, NCA-90, Ia, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES, TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAIL receptor R1, TRAIL receptor R2, VEGFR, EGFR, P1GF, complement factor C3, C3a, C3b, C5a, C5, and an oncogene product. 
     
     
         3 . The method of  claim 1 , wherein the antibody or antibody fragment is selected from the group consisting of an IgG, a F(ab′) 2 , a F(ab) 2 , a Fab′, a Fab, a Fv, a scFv and a dAb. 
     
     
         4 . The method of  claim 1 , wherein the antibody or antibody fragment is selected from the group consisting of hR1 (anti-IGF-1R), hPAM4 (anti-mucin), hA20 (anti-CD20), hLL1 (anti-CD74), hLL2 (anti-CD22), hMN-14 (anti-CEA), hMN-15 (anti-CEA) and hRS7 (anti-EGP-1). 
     
     
         5 . The method of  claim 1 , wherein the complex comprises four copies of the toxin. 
     
     
         6 . The method of  claim 1 , wherein the toxin is ranpirnase. 
     
     
         7 . The method of  claim 1 , wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, B cell lymphoma, B cell leukemia, T cell lymphoma, T cell leukemia, acute lymphoid leukemia, chronic lymphoid leukemia, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, carcinoma, melanoma, sarcoma, glioma, skin cancer, oral cavity cancer, gastrointestinal tract cancer, colon cancer, stomach cancer, pulmonary tract cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, urinary bladder cancer, pancreatic cancer, bone cancer, liver cancer, gall bladder cancer, kidney cancer, and testicular cancer. 
     
     
         8 . The method of  claim 1 , wherein the cancer is selected from the group consisting of cervical, breast, prostate, lung adenocarcinoma, pancreatic and ovarian cancer. 
     
     
         9 . The method of  claim 1 , further comprising administering to the subject a therapeutic agent selected from the group consisting of another anti-TAA antibody, a cytotoxic agent, an immunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, an antibiotic, a hormone, a hormone antagonist, a cytokine, a chemokine, a drug, a prodrug, and an enzyme. 
     
     
         10 . The method of  claim 9 , wherein the therapeutic agent is selected from the group consisting of 5-fluorouracil, aplidin, azaribine, anastrozole, an anthracycline, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, a Cox-2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, cladribine, a camptothecan, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, an estrogen receptor binding agent, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and a  vinca  alkaloid. 
     
     
         11 . The method of  claim 9 , wherein the chemokine is selected from the group consisting of RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10. 
     
     
         12 . The method of  claim 9 , wherein the immunomodulator is 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, tumor necrosis factor (TNF), interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α, interferon-β, interferon-γ, and S1 factor. 
     
     
         13 . The method of  claim 12 , wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α.alpha. tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin, NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), interferon-α, interferon-β, interferon-γ, macrophage-CSF (M-CSF), interleukin-1 (IL-1), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.