Glycan-based antibody-drug conjugates

Abstract

Genetically engineered antibodies containing non-native N-glycosylated sites, preparation of the antibodies in yeast and fungi, site-specific conjugation of drugs to these antibodies, and methods of treatment utilizing these antibodies are described herein.

We claim: 
     
         1 . An engineered IgG antibody or heavy chain constant domain fragment comprising
 an S134N mutation in the heavy chain constant domain, which forms a first non-native N-glycosylation site having the amino acid sequence NTS over positions 134-136 of the heavy chain constant domain and a G161T or G161S mutation, which forms a second non-native N-glycosylation site in the heavy chain constant domain having the amino acid sequence NST over positions 159-161 of the heavy chain constant domain, said non-native N-glycosylation sites having an N-glycan attached to the N at position 134 and 159, wherein the N-glycans have a Gal 2 GlcNAc 2 Man 3 GlcNAc 2  or GalGlcNAcMan 3 GlcNAc 2  glycoform in which the terminal galactose residues have been oxidized to a C-6 aldehyde group, which is conjugated to a reactive amine group of a derivatized drug by oxime bonds, and wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering.   
     
     
         2 . An engineered IgG antibody or heavy chain constant domain fragment of  claim 1 , wherein N-glycan comprises a Gal 2 GlcNAc 2 Man 3 GlcNAc 2  glycoform. 
     
     
         3 . An engineered IgG antibody or heavy chain constant domain fragment of  claim 1 , wherein N-glycan comprises a Gal 2 GlcNAc 2 Man 3 GlcNAc 2  glycoform. 
     
     
         4 . An engineered IgG antibody or heavy chain constant domain fragment of  claim 1 , wherein the engineered IgG antibody or heavy chain constant domain fragment further comprises one to ten amino acid mutations or pairs of mutations are selected from the group consisting of N203 T, N203S, V363T, V363S, Q438N, S176N, A118N, S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T, Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S, Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T, and G194N/Q196S, wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering. 
     
     
         5 . An engineered IgG antibody or heavy chain constant domain fragment of  claim 1 , wherein the derivatized drug is selected from the group consisting of a polymer, cytotoxic agent, a radionuclide, fluorescent or chemiluminescent labels, steroid, steroid receptor agonist, signal transduction inhibitor, a peptide and scFv. 
     
     
         6 . An engineered IgG antibody or heavy chain constant domain fragment of  claim 1 , wherein the derivatized drug comprises a cytotoxic agent. 
     
     
         7 . The engineered IgG antibody or antigen binding fragment of  claim 1 , wherein the IgG antibody further comprises one or two N-glycosylation sites in the variable domain that have been generated by amino acid mutations selected from the group consisting of Q105N and S113N, wherein the numbering is according to the Kabat numbering system. 
     
     
         8 . The engineered IgG antibody or constant domain fragment of  claim 1 , wherein the IgG antibody is selected from the group consisting of anti-Her2, anti-Her2/neu, anti-glycoprotein IIb/IIIa, anti-TNF-α, anti-CD52, anti-CD25, anti-BAFF, anti-Vascularendothelial growth factor, anti-CD30, anti-IL-1β, anti-epidermal growth factor receptor, anti-RANK Ligand, anti-Complement C5, anti-CD11a, anti-CD33, anti-CD20, anti-CTLA-4, anti-T cell CD3 Receptor, anti-α-4 (α4) integrin, anti, anti-Immunoglobulin E, anti-RSV F protein, anti-epidermal growth factor receptor, anti-VEGF-A, anti-ErbB2, anti-IL-12/IL-23, anti-integrin α4β7, anti-CD274, anti-3-amyloid, anti-4-1BB, anti-SAC, anti-5T4, anti-ACVR2B, anti-adenocarcinomaantigen, anti-AGS-22M6, anti-α-fetoprotein, anti-angiopoietin 2, anti-angiopoietin 3, anti-anthrax toxin, anti-AOC3, anti-, anti-B7-H3, anti- Bacillus  anthracia, anti-β amyloid, anti-B-lymphoma cell, anti-C242 antigen, anti-05, anti-CA-125, anti-carbonic anhydrase 9, anti-cardiac myosin, anti-CCL11, anti-CCR4, anti-CCR5, anti-CD11/CD18, anti-CD125, anti-CD140a, anti-CD147, anti-CD15, anti-CD152, anti-CD154, anti-CD19, anti-CD2, anti-CD200, anti-CD22, anti-CD221, anti-CD23, anti-CD27, anti-CD28, anti-CD3, anti-CD3 epsilon, anti-CD30, anti-CD37, anti-CD38, anti-CD4, anti-CD40, anti-CD41, anti-CD44, anti-CD5, anti-CD51, anti-CD52, anti-CD56, anti-CD6, anti-CD70, anti-CD74, anti-CD79B, anti-CD80, anti-CEA, anti-CFD, anti-ch4D5, anti-CLDN18.2, anti- C. difficile , anti-clumping factor A, anti-CSF2, anti-cytomegalovirus, anti-CMV gp B, anti-DLL4, anti-DRS, anti- E. coli  shiga toxin type-1 or 2, anti-EGFL7, anti-endotoxin, anti-EpCAM, anti-EpCAM/CD3, anti-episialin, anti-ERBB3, anti- Escherichia coli , anti-F protein, anti-FAP, anti-fibrin II, anti-βchain, anti-fibronectin extra domain-B, anti-folate receptor 1, anti-Frizzled receptor, anti-GD2 ganglioside, anti-GD3 ganglioside, anti-GMCSF receptor α-chain, anti-GPNMB, anti-Influenza, anti-Influenza hemagglutinin, anti-hepatitis B, anti-surface antigen, anti-HER1, anti-HER3, anti-HGF, anti-HHGFR, anti-HIV-1, anti-HLA-DR, anti-HNGF, anti-Hsp90, anti-human scatter factor receptor kinase, anti-human TNF, anti-human β-amyloid, anti-CD54, anti-IFN-α, anti-IFN-γ, anti-IgE Fc region, anti-IGF-1 receptor, anti-IGF-I, anti-IgG4, anti-IGHE, anti-IL-13, anti-IL-17, anti-IL-17A, anti-IL-10, anti-IL-22, anti-IL-23, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-6 receptor, anti-IL9, anti-ILGF2, anti-insulin-like growth factor I receptor anti-integrin α4, anti-integrin α5β1, anti-integrin α7β7, anti-integrin αIIbβ3, anti-integrin αvβ3, anti-interferon receptor, anti-interferon α/β receptor, anti-interferon γ-induced protein, anti-ITGA2, anti-KIR2D, anti-Lewis-Y antigen, anti-lipoteichoic acid, anti-LOXL2, anti-L-selectin (CD62L), anti-LTA, anti-MCP-1, anti-mesothelin, anti-MS4A1, anti-MUC1, anti-mucin CanAg, anti-myostatin, anti-NARP-1, anti-NCA-90, anti-NGF, anti-N-glycolylneuraminic acid, anti-NOGO-A, anti-Notch receptor, anti-NRP1, anti-Oryctolagus  cuniculus , anti-OX-40, anti-oxLDL, anti-PCSK9, anti-PD-1, anti-PDCD1, anti-PDGF-R α, anti-phosphate-sodium co-transporter, anti-phosphatidylserine, anti-prostatic carcinoma cells, anti- Pseudomonas aeruginosa , anti-rabies virus, anti-rabies virus glycoprotein, anti-respiratory syncytial virus, anti-RHD, anti-Rhesus factor, anti-RON, anti-RTN4, anti-sclerostin, anti-SDC1, anti-selectin P, anti-SLAMF7, anti-SOST, anti-sphingosine-1-phosphate, anti-TAG-72, anti-T-cell receptor, anti-TEM1, anti-tenascin C, anti-TFPI, anti-TGFβ1, anti-TGFβ2, anti-TGF-β, anti-TRAIL-R1, anti-TRAIL-R2, anti-tumor antigen CTAA16.88, anti-TWEAK receptor, anti-TYRP1, anti-VEGF-A, anti-VEGFR-1, anti-VEGFR2, anti-vimentin, anti-VWF, anti-IL-1, anti-IL-2, anti-IL-5, anti-IL-8, anti-IL-12, anti-IL-15, anti-IL-18, anti-IL-20, anti-IL-21, anti-IL-23R, anti-IL-25, anti-IL-27, anti-IL-33, anti-CD14, anti-CD18, anti-CD64, anti-CD200, anti-CD200R, anti-TSLP, anti-TSLPR, anti-PDL1, anti-VLA-4, anti-E-selectin, anti-Fact II, anti-ICAM-3, anti-β2-integrin, anti-CBL, anti-LCAT, anti-CR3, anti-MDL-1, anti-GITR, anti-CGRP, anti-TRKA, anti-IGF1R, and anti-GTC. 
     
     
         9 . The engineered IgG antibody or constant domain fragment of  claim 1 , wherein the IgG antibody is selected from the group consisting of abciximab, adalimumab, certolizumab pegol, golimumab, infliximab, alemtuzumab, basiliximab), belimumab, bevacizumab, brentuximab, canakinumab, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, ipilimumab, muromonab-cd3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, atlizumab, tositumomab, trastuzumab, ustekinumab, and vedolizumab. 
     
     
         10 . A method of preparing a conjugated N-glycosylated IgG antibody or fragment thereof comprising an N-glycan attached to the N at position 134, wherein the N-glycan has a Gal 2 GlcNAc 2 Man 3 GlcNAc 2  or GalGlcNAcMan 3 GlcNAc 2  glycoform in which the terminal galactose residues are conjugated to a reactive amine group of a derivatized drug by an oxime bond, the method comprising:
 (a) transforming a yeast or filamentous fungus host cell genetically engineered to produce N-glycans comprising terminal galactose residues of the structure Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2  or the structure Gal (1-2) GlcNAc (1-2) Man 5 GlcNAc 2  with a nucleic acid encoding an IgG heavy chain constant domain having the amino acid sequence NTS over positions 134-136 of the heavy chain constant domain;   b) culturing the transformed host cell under conditions that allow the expression of the IgG heavy chain constant domain comprising terminal galactose residues;   (c) contacting the expressed IgG heavy chain constant domain with a reagent that oxidizes the terminal galactose residues to a C-6 aldehyde group; and   (d) conjugating the reactive amine group of a derivatized drug to the C-6 aldehyde group to produce the conjugated N-glycosylated IgG antibody or fragment thereof, wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering.   
     
     
         11 . The method of  claim 10 , wherein the IgG heavy chain further comprising a G161 T mutation, which forms a second non-native N-glycosylation site in the heavy chain constant domain having the amino acid sequence NST over positions 159-161 of the heavy chain constant domain, wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering. 
     
     
         12 . The method of  claim 10 , wherein the engineered IgG antibody or heavy chain constant domain fragment further comprises one to ten amino acid mutations or pairs of mutations are selected from the group consisting of N203 T, N203 S, V363T, V363S, Q438N, S176N, A118N, S132N, K133N, A162N, T195N, K2l10T, Y391T, F423T, F423S, Y436T, Y436S, L193N, K392T, K392S, F423 T, S176N/G178T, S176N/G178S, Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T, and G194N/Q196S, whereinthe amino acid positions of the heavy chain contant domain are according to Eu numbering. 
     
     
         13 . The method of  claim 10 , wherein the nucleic acid encodes an IgG antibody and further comprises one or two N-glycosylation sites in the variable domain that have been generated by amino acid mutations selected from the group consisting of Q105N and S113N, wherein the numbering is according to the Kabat numbering system. 
     
     
         14 . The method of  claim 11 , wherein the engineered IgG antibody or heavy chain constant domain fragment further comprises one to ten amino acid mutations or pairs of mutations are selected from the group consisting of N203 T, N203 S, V363T, V363S, Q438N, S176N, A118N, S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T, Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S, Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T, andG194N/Q196S, wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering. 
     
     
         15 . The method of  claim 11 , wherein the nucleic acid encodes an IgG antibody and further comprises one or two N-glycosylation sites in the variable domain that have been generated by amino acid mutations selected from the group consisting of Q105N and S113N, wherein the numbering is according to the Kabat numbering system. 
     
     
         16 . The method of  claim 10 , wherein the yeast host cell is selected from the group consisting of  Pichia pastoris  ( Komagataella pastoris ),  Pichia finlandica , Pichia trehalophila,  Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae , Pichiathermotolerans, Pichia  salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia minuta  ( Ogataea minuta, Pichia lindneri ), Pichia sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula  polymorphs,  Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae , Trichodermareesei,  Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum , and  Neurospora crassa.    
     
     
         17 . An engineered IgG antibody or heavy chain constant domain fragment comprising
 a G161 T mutation, which forms a second non-native N-glycosylation site in the heavy chain constant domain having the amino acid sequence NST over positions 159-161 of the heavy chain constant domain, said non-native N-glycosylation site having an N-glycan attached to the N at position 134, wherein the N-glycan has a Gal 2 GlcNAc 2 Man 3 GlcNAc 2  glycoform or GalGlcNAcMan 3 GlcNAc 2  glycoform in which the terminal galactose residues have been oxidized to a C-6 aldehyde group, which is conjugated to a reactive amine group of a derivatized drug by an oxime bond, and wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering.   
     
     
         18 . The engineered IgG antibody or constant domain fragment of  claim 17 , wherein the engineered IgG antibody or heavy chain constant domain fragment further comprises one to ten amino acid mutations or pairs of mutations are selected from the group consisting of N203 T, N203S, V363T, V363S, Q438N, S176N, A118N, S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T, Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S, Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T, and G194N/Q196S, wherein the amino acid positions of the heavy chain contant domain are according to Eu numbering. 
     
     
         19 . The engineered IgG antibody or constant domain fragment of  claim 17 , wherein the IgG antibody and further comprises one or two N-glycosylation sites in the variable domain that have been generated by amino acid mutations selected from the group consisting of Q105N and S113N, wherein the numbering is according to the Kabat numbering system. 
     
     
         20 . The engineered IgG antibody or constant domain fragment of  claim 17 , wherein the IgG antibody is
 (a) selected from the group consisting of anti-Her2, anti-Her2/neu, anti-glycoprotein IIb/IIIa, anti-TNF-α, anti-CD52, anti-CD25, anti-BAFF, anti-Vascularendothelial growth factor, anti-CD30, anti-IL-1β, anti-epidermal growth factor receptor, anti-RANK Ligand, anti-Complement C5, anti-CD11a, anti-CD33, anti-CD20, anti-CTLA-4, anti-T cell CD3 Receptor, anti-α-4 (a4) integrin, anti, anti-Immunoglobulin E, anti-RSV F protein, anti-epidermal growth factor receptor, anti-VEGF-A, anti-ErbB2, anti-IL-12/IL-23, anti-integrin α407, anti-CD274, anti-3-amyloid, anti-4-1BB, anti-SAC, anti-5T4, anti-ACVR2B, anti-adenocarcinomaantigen, anti-AGS-22M6, anti-α-fetoprotein, anti-angiopoietin 2, anti-angiopoietin 3, anti-anthrax toxin, anti-AOC3, anti-, anti-B7-H3, anti- Bacillus anthracia , anti-β amyloid, anti-B-lymphoma cell, anti-C242 antigen, anti-05, anti-CA-125, anti-carbonic anhydrase 9, anti-cardiac myosin, anti-CCL11, anti-CCR4, anti-CCR5, anti-CD11/CD18, anti-CD125, anti-CD140a, anti-CD147, anti-CD15, anti-CD152, anti-CD154, anti-CD19, anti-CD2, anti-CD200, anti-CD22, anti-CD221, anti-CD23, anti-CD27, anti-CD28, anti-CD3, anti-CD3 epsilon, anti-CD30, anti-CD37, anti-CD38, anti-CD4, anti-CD40, anti-CD41, anti-CD44, anti-CD5, anti-CD51, anti-CD52, anti-CD56, anti-CD6, anti-CD70, anti-CD74, anti-CD79B, anti-CD80, anti-CEA, anti-CFD, anti-ch4D5, anti-CLDN18.2, anti- C. difficile , anti-clumping factor A, anti-CSF2, anti-cytomegalovirus, anti-CMV gp B, anti-DLL4, anti-DRS, anti- E. coli  shiga toxin type-1 or 2, anti-EGFL7, anti-endotoxin, anti-EpCAM, anti-EpCAM/CD3, anti-episialin, anti-ERBB3, anti- Escherichia coli , anti-F protein, anti-FAP, anti-fibrin II, anti-β chain, anti-fibronectin extra domain-B, anti-folate receptor 1, anti-Frizzled receptor, anti-GD2 ganglioside, anti-GD3 ganglioside, anti-GMCSF receptor α-chain, anti-GPNMB, anti-Influenza, anti-Influenza hemagglutinin, anti-hepatitis B, anti-surface antigen, anti-HER1, anti-HER3, anti-HGF, anti-HHGFR, anti-HIV-1, anti-HLA-DR, anti-HNGF, anti-Hsp90, anti-human scatter factor receptor kinase, anti-human TNF, anti-human β-amyloid, anti-CD54, anti-IFN-α, anti-IFN-γ, anti-IgE Fc region, anti-IGF-1 receptor, anti-IGF-I, anti-IgG4, anti-IGHE, anti-IL-13, anti-IL-17, anti-IL-17A, anti-IL-10, anti-IL-22, anti-IL-23, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-6 receptor, anti-IL9, anti-ILGF2, anti-insulin-like growth factor I receptor, anti-integrin α4, anti-integrin α5β1, anti-integrin α7β7, anti-integrin αIIbβ3, anti-integrin αvβ3, anti-interferon receptor, anti-interferon α/β receptor, anti-interferon 7-induced protein, anti-ITGA2, anti-KIR2D, anti-Lewis-Y antigen, anti-lipoteichoic acid, anti-LOXL2, anti-L-selectin (CD62L), anti-LTA, anti-MCP-1, anti-mesothelin, anti-MS4A1, anti-MUC1, anti-mucin CanAg, anti-myostatin, anti-NARP-1, anti-NCA-90, anti-NGF, anti-N-glycolylneuraminic acid, anti-NOGO-A, anti-Notch receptor, anti-NRP1, anti-Oryctolagus  cuniculus , anti-OX-40, anti-oxLDL, anti-PCSK9, anti-PD-1, anti-PDCD1, anti-PDGF-R α, anti-phosphate-sodium co-transporter, anti-phosphatidylserine, anti-prostatic carcinoma cells, anti- Pseudomonas aeruginosa , anti-rabies virus, anti-rabies virus glycoprotein, anti-respiratory syncytial virus, anti-RHD, anti-Rhesus factor, anti-RON, anti-RTN4, anti-sclerostin, anti-SDC1, anti-selectin P, anti-SLAMF7, anti-SOST, anti-sphingosine-1-phosphate, anti-TAG-72, anti-T-cell receptor, anti-TEM1, anti-tenascin C, anti-TFPI, anti-TGFβ1, anti-TGFβ2, anti-TGF-β, anti-TRAIL-R1, anti-TRAIL-R2, anti-tumor antigen CTAA16.88, anti-TWEAK receptor, anti-TYRP1, anti-VEGF-A, anti-VEGFR-1, anti-VEGFR2, anti-vimentin, anti-VWF, anti-IL-1, anti-IL-2, anti-IL-5, anti-IL-8, anti-IL-12, anti-IL-15, anti-IL-18, anti-IL-20, anti-IL-21, anti-IL-23R, anti-IL-25, anti-IL-27, anti-IL-33, anti-CD14, anti-CD18, anti-CD64, anti-CD200, anti-CD200R, anti-TSLP, anti-TSLPR, anti-PDL1, anti-VLA-4, anti-E-selectin, anti-Fact II, anti-ICAM-3, anti-02-integrin, anti-CBL, anti-LCAT, anti-CR3, anti-MDL-1, anti-GITR, anti-CGRP, anti-TRKA, anti-IGF1R, and anti-GTC; or   (b) selected from the group consisting of abciximab, adalimumab, certolizumab pegol, golimumab, infliximab, alemtuzumab, basiliximab), belimumab, bevacizumab, brentuximab, canakinumab, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, ipilimumab, muromonab-cd3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, atlizumab, tositumomab, trastuzumab, ustekinumab, and vedolizumab.