Pro-drug form (p2pdox) of the highly potent 2-pyrrolinodoxorubicin conjugated to antibodies for targeted therapy of cancer

Abstract

Disclosed are methods, compositions and uses of conjugates of prodrug forms of 2-pyrrolinodoxorubicin (P2PDox) with antibodies or antigen-binding fragments thereof (ADCs), with targetable construct peptides or with other targeting molecules that are capable of delivering the P2PDox to a targeted cell, tissue or pathogen. Once delivered to the target cell, the ADC or peptide conjugate is internalized, a highly toxic 2-pyrrolinodoxorubicin (2-PDox) is released intracellularly. The P2PDox-peptide or ADC conjugates are of use to treat a wide variety of diseases, such as cancer, autoimmune disease or infectious disease.

What is claimed is: 
     
         1 . A composition comprising an antibody, antigen-binding antibody fragment, targeting peptide, or non-antibody cell-targeting construct attached to pro-2-pyrrolinodoxorubicin (P2PDox). 
     
     
         2 . The composition of  claim 1 , further comprising an aqueous medium. 
     
     
         3 . The composition of  claim 1 , further comprising a buffer. 
     
     
         4 . The composition of  claim 4 , wherein the buffer is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (IVIES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 1,4-piperazinediethanesulfonic acid (PIPES), and phosphate. 
     
     
         5 . The composition of  claim 1 , further comprising at least one excipient selected from the group consisting of trehalose and polysorbate 80. 
     
     
         6 . The composition of  claim 1 , further comprising a salt selected from the group consisting of NaCl, KCl, MgCl 2 , and CaCl 2 . 
     
     
         7 . The composition of  claim 1 , wherein the antibody, antigen-binding antibody fragment, targeting peptide, or non-antibody cell-targeting construct binds to a tumor-associated antigen. 
     
     
         8 . The composition of  claim 1 , wherein the antibody, antigen-binding antibody fragment, targeting peptide, or non-antibody cell-targeting construct binds to an antigen selected from the group consisting of CTLA4, PD1, PD-L1, 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, CD47, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, c-MET, B7, ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GRO-β, histone H2B, histone H3, histone H4, HMGB-1, hypoxia inducible factor (HIF), HM1.24, 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-23, IL-25, IP-10, LIV-1, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, NCA-95, NCA-90, Ia, HM1.24, EGP-1 (TROP-2), EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAIL receptor R1, TRAIL receptor R2, VEGFR, EGFR, PlGF, complement factors C3, C3a, C3b, C5a, C5, and an oncogene product. 
     
     
         9 . The composition of  claim 1 , wherein the non-antibody cell-targeting construct is selected from the group consisting of an avimer, a fynomer, a phage display peptide, an aptamer, an affibody and a nanobody. 
     
     
         10 . The composition of  claim 1 , wherein the antibody is selected from the group consisting of hR1 (anti-IGF-1R), hPAM4 (anti-MUC5ac), hA20 (anti-CD20), GA101 (anti-CD20), hA19 (anti-CD19), hIMMU-31 (anti-AFP), hLL1 (anti-CD74), hLL2 (epratuzumab, anti-CD22), hRFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hL243 IgG4P (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1 or anti-TROP-2), hMN-3 (anti-CEACAM6), Ab124 (anti-CXCR4) and Ab125 (anti-CXCR4). 
     
     
         11 . The composition of  claim 1 , wherein the antibody fragment is selected from the group consisting of F(ab′) 2 , F(ab) 2 , Fab, Fab′ and scFv fragments. 
     
     
         12 . The composition of  claim 1 , wherein the antibody or fragment thereof comprises human constant regions selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 
     
     
         13 . The composition of  claim 1 , wherein the antibody is a non-G1m1 (nG1m1) antibody. 
     
     
         14 . The composition of  claim 1 , wherein the antibody has a G1m3 heavy chain allotype. 
     
     
         15 . The composition of  claim 1 , wherein the antibody has a nG1m1,2 heavy chain null allotype. 
     
     
         16 . The composition of  claim 1 , wherein the antibody has a Km3 light chain allotype. 
     
     
         17 . The composition of  claim 1 , wherein the antibody comprises heavy chain constant region amino acid residues arginine-214, glutamic acid-356, methionine-358 and alanine-431. 
     
     
         18 . The composition of  claim 1 , wherein the antibody is selected from the group consisting of a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, a bispecific antibody, a multispecific antibody, and an antibody fusion protein. 
     
     
         19 . The composition of  claim 1 , wherein the targeting peptide is selected from the group consisting of IMP402, IMP513, IMP514, IMP515 and IMP516. 
     
     
         20 . The composition of  claim 1 , wherein the P2PDox is attached to the antibody, antibody fragment or peptide by a cross-linker selected from the group consisting of SMCC hydrazide, aminoxy, phenylhydrazide and 4-(hydrazinosulfonyl)benzoic acid. 
     
     
         21 . The composition of  claim 1 , wherein the P2PDox comprises a maleimide moiety and is attached to the antibody, antibody fragment or peptide by reaction of the maleimide with a cysteine residue. 
     
     
         22 . The composition of  claim 1 , wherein the P2PDox forms intramolecular cross-links with the antibody or antigen-binding antibody fragment. 
     
     
         23 . The composition of  claim 22 , wherein the intramolecular cross-links stabilize the composition in vivo and prevent release of free drug in circulation. 
     
     
         24 . The composition of  claim 1 , wherein the targeting peptide comprises at least one hapten moiety selected from the group consisting of In-DTPA and HSG. 
     
     
         25 . The composition of  claim 1 , further comprising at least one therapeutic agent selected from the group consisting of a drug, a prodrug, an enzyme, a tyrosine kinase inhibitor, a sphingosine inhibitor, an immunomodulator, a cytokine, a hormone, a second antibody, a second antibody fragment, an immunoconjugate, an anti-angiogenic agent, a pro-apoptosis agent and a cytotoxic agent. 
     
     
         26 . The composition of  claim 25 , wherein the drug is selected from the group consisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epipodophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839. 
     
     
         27 . The composition of  claim 25 , wherein the immunoconjugate comprises a toxin attached to an antibody or antigen-binding antibody fragment and selected from the group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), ranpirnase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,  Pseudomonas  exotoxin, and  Pseudomonas  endotoxin. 
     
     
         28 . The composition of  claim 25 , wherein the immuoconjugate comprises a radionuclide attached to an antibody or antigen-binding antibody fragment and selected from the group consisting of  111 In,  111 At,  177 Lu,  211 Bi,  212 Bi,  213 Bi,  211 At,  62 Cu,  67 Cu,  90 Y,  125 I,  131 I,  133 I,  32 P,  33 P,  47 Sc,  111 Ag,  67 Ga,  153 Sm,  161 Tb,  152 Dy,  166 Dy,  161 Ho,  166 Ho,  186 Re,  188 Re,  189 Re,  211 Pb,  212 Pb,  223 Ra,  225 Ac,  77 As,  89 Sr,  99 Mo,  105 Rh,  149 Pm,  169 Er,  194 Ir,  58 Co,  80m Br,  99m Tc,  103m Rh,  109 Pt,  119 Sb,  189m Os,  192 Ir  219 Rn,  215 Po,  221 Fr,  255 Fm,  11 C,  13 N,  15 O,  75 Br,  198 Au,  199 Au,  224 Ac,  77 Br,  113m In,  95 Ru,  97 Ru,  103 Ru,  105 Ru,  107 Hg,  203 Hg,  121m Te,  122m Te,  125m Te,  227 Th,  165 Tm,  167 Tm,  168 Tm,  197 Pt,  109 Pd,  142 Pr,  143 Pr,  161 Tb,  57 Co,  58 Co,  51 Cr,  59 Fe,  75 Se,  201 Tl,  76 Br and  16 Yb. 
     
     
         29 . The composition of  claim 25 , wherein the immunomodulator is selected from the group consisting of a cytokine, a chemokine, a stem cell growth factor, a lymphotoxin, an hematopoietic factor, a colony stimulating factor (CSF), an interferon, erythropoietin, thrombopoietin, a tumor necrosis factor (TNF), an interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF) and a stem cell growth factor designated “S1 factor”. 
     
     
         30 . The composition of  claim 25 , wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, tumor necrosis factor-.alpha., tumor necrosis factor-.beta., mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), NGF-.beta., platelet-growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferon-α, interferon-β, interferon-γ, macrophage-CSF (M-CSF), IL-1, IL-la, 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, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and lymphotoxin.