US20150104468A1 - Site-specific labeling methods and molecules produced thereby - Google Patents

Site-specific labeling methods and molecules produced thereby Download PDF

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US20150104468A1
US20150104468A1 US14/402,808 US201314402808A US2015104468A1 US 20150104468 A1 US20150104468 A1 US 20150104468A1 US 201314402808 A US201314402808 A US 201314402808A US 2015104468 A1 US2015104468 A1 US 2015104468A1
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nhc
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antibody
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Bernhard Hubert GEIERSTANGER
Jan Grunewald
Badry Bursulaya
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Novartis AG
Novartis Institutes for Biomedical Research Inc
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IRM LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A61K47/48715—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • the present invention relates to site-specific labeling process and molecules produced thereby.
  • Conjugation has been widely used to optimize the properties of biologically active proteins, such as protein therapies, antibody drug conjugates (ADCs), vaccines, tissue selective targeting vehicles, molecular diagnostics, and protein nucleic acid conjugates.
  • Traditional conjugation method utilizes lysine based covalent ligation, which makes it difficult to achieve homogeneity due to the abundance of lysines on the protein's surface.
  • Site-specific labeling of proteins can be achieved by post-translational enzymatic reactions, for example, using human O 6 -alkylguanine-DNA alkyl-transferase (AGT), biotin ligase, transglutaminase, sortase, cutinase, or 4′-phosphopantetheinyl transferases for the covalent attachment of a label to a protein.
  • AGT human O 6 -alkylguanine-DNA alkyl-transferase
  • biotin ligase transglutaminase
  • sortase sortase
  • cutinase or 4′-phosphopantetheinyl transferases
  • the AGT is fused to a target protein of interest, followed by the addition of a labeled O 6 -benzylguanine, which is a suicide substrate for the AGT (Keppler et al., Nat. Biotechnol. 21:86-89, 2003).
  • This approach is the basis for a technology called SNAP-TagTM, which utilizes a 180 amino acid tag (Tirat et al., International Journal of Biological Macromolecules, 39:66-76, 2006).
  • labeling of proteins using this approach occurs only at the C- or N-termini.
  • biotin protein ligase attaches biotin to the biotin carrier domain of certain carboxylases or decarboxylases.
  • BPL catalyzes, in a two-step, adenosine-5′-triphosphate (ATP)-dependent reaction, the post-translational formation of an amide bond between the carboxyl group of biotin and the ⁇ -amino group of a specific lysine residue located within a highly conserved Ala-Met-Lys-Met (SEQ ID NO: 1017) recognition located motif within the biotin carrier domain (Tirat et al., International Journal of Biological Macromolecules, 39:66-76, 2006).
  • ATP adenosine-5′-triphosphate
  • This approach can be used to create fusion tags at the C-terminus, the N-terminus or even within the target protein and is the basis for a technology called BioEaseTM (72 amino acid tag) and AviTagTM (uses the biotin ligase, BirA and 15-residue acceptor peptide tag (AP)).
  • BioEaseTM 72 amino acid tag
  • AviTagTM uses the biotin ligase, BirA and 15-residue acceptor peptide tag (AP)
  • Transglutaminases catalyze the formation of stable isopeptidic bonds between the side chains of glutamine (Gln) and lysine (Lys) with the loss of ammonia, and have been used to label glutamine side chains in proteins with fluorophores in vitro (Sato et al., Biochemistry 35:13072-13080, 1996). Also, bacterial and human tissue transglutaminases (BTGase and TG2) have been used to catalyze the post-translational modification of different IgG's via the Lys or Gln side chains located in the IgG heavy chain (Mindt et al., Bioconjugate Chem. 19:271-278, 2008; Jeger et al., Angew. Chem. Int. 49:9995-9997, 2010).
  • Sortases have been used for C-terminal and N-terminal site specific modification of proteins, where sortase A catalyzes the transpeptidation reaction (Antos et al., JACS, 131:10800-10801, 2009).
  • Cutinase is a 22-kDa serine esterase that forms a site-specific covalent adduct with phosphonate ligands that is resistant to hydrolysis. Cutinases have been used for C-terminal and N-terminal site specific modification of antibodies followed by immobilization onto surfaces (Kwon et al., Anal. Chem. 76:5713-5720, 2004; Hodneland et al., Proc. Natl. Acad. Sci. U.S.A., 99:5048-5052, 2002).
  • acyl carrier proteins ACPs
  • PCPs peptidyl carrier proteins
  • PKSs polyketide synthases
  • NRPSs nonribosomal peptide synthetases
  • carrier proteins Due to the comparably small size of the carrier proteins and the ability of 4′-phosphopantetheinyl transferases to accept functionalized CoA analogues as substrates, researchers have used carrier proteins as fusion tags to label target proteins with a variety of small molecule probes (see, e.g., La Clair et al., Chem. Biol. 11(2):195-201, 2004; Yin et al., J. Am. Chem. Soc. 126(25):7754-7755, 2004).
  • Walsh and co-workers used phage display to identify 8- to 12-residue peptides that are recognized as efficient substrates by the bacterial 4′-phosphopantetheinyl transferase Sfp (previously identified as a genetic locus responsible for surfactin production) and AcpS (Yin et al., Proc. Natl. Acad. Sci. USA 102(44):15815-15820, 2005; Zhou et al., ACS Chem. Biol. 2(5):337-346, 2007; Zhou et al., J. Am. Chem. Soc. 130(30):9925-9930, 2008).
  • ADCs Antibody drug conjugates
  • ADCs have been used for the local delivery of cytotoxic agents in the treatment of cancer (see e.g., Lambert, Curr. Opinion In Pharmacology 5:543-549, 2005).
  • ADCs allow targeted delivery of the drug moiety where maximum efficacy with minimal toxicity may be achieved.
  • the present invention provides modified antibodies or fragments thereof, which comprise at least one peptide tag that is a substrate of 4′-phosphopantetheinyl transferase, and is located within the structural loop of said antibodies or antibody fragments.
  • the present invention further provides immunoconjugates comprising such modified antibodies or antibody fragments, and a terminal group.
  • the present invention also provides methods of making such modified antibodies, antibody fragments, and the immunoconjugates, as well as methods of using such compositions.
  • the present invention provides modified antibodies or fragments thereof, which comprise at least one peptide tag that is a substrate of 4′-phosphopantetheinyl transferase, and is located within the structural loop of said antibodies or antibody fragments, and wherein the 4′-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima PPTase, human PPTase or a mutant or homolog form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • the peptide tag is selected from the group consisting of: GDSLSWLLRLLN (SEQ ID NO: 1), GDSLSWL (SEQ ID NO: 2), GDSLSWLVRCLN (SEQ ID NO: 3), GDSLSWLLRCLN (SEQ ID NO: 4), GDSLSWLVRLLN (SEQ ID NO: 5), GDSLSWLLRSLN (SEQ ID NO: 6), GSQDVLDSLEFIASKLA (SEQ ID NO: 7), VLDSLEFIASKLA (SEQ ID NO: 8), DSLEFIASKLA (SEQ ID NO: 9), GDSLDMLEWSLM (SEQ ID NO: 10), GDSLDMLEWSL (SEQ ID NO: 11), GDSLDMLEWS (SEQ ID NO: 12), GDSLDMLEW (SEQ ID NO: 13), DSLDMLEW (SEQ ID NO: 14), GDSLDM (SEQ ID NO: 15), LDSVRMMALAAR (SEQ ID NO: 16), LD
  • the present invention provides modified antibodies or fragments thereof, which comprise at least one peptide tag that is a substrate of 4′-phosphopantetheinyl transferase, and is located within the structural loop of VH, VL, CH1, CH2, CH3, or C L region of the antibody or fragment thereof.
  • the peptide tag is inserted between any two amino acids that are listed in Table 1.
  • the present invention provides modified antibodies or antibody fragments comprising at least one peptide tag that is a substrate of 4′-phosphopantetheinyl transferase, and is located within the structural loop of the CH1 region of an antibody or fragment thereof.
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the peptide tag is inserted between amino acid residues 2 and 3 of the V H or V L domain, or between amino acid residues 63 and 64 of the V H domain, or between 64 and 65 of the V H domain, or between 138 and 139 of the CH1 domain, or between 197 and 198 of the CH1 domain, or between 359 and 360 of the CH3 domain, or between 388 and 389 of the CH3 domain, or after 447 of the CH3 domain of a parental antibody or fragment thereof.
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the peptide tag is inserted between amino acid residues 2 and 3 of the VH or VL domain, or between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 162 and 163, or between 164 and 165, or between 165 and 166, or between 194 and 195, or between 195 and 196 of the CH1 domain, or between 388 and 389, or between 445 and 446, or between 446 and 447 of the CH3 domain of a parental antibody or fragment thereof.
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the peptide tag is inserted between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 162 and 163, or between 164 and 165, or between 165 and 166, or between 194 and 195, or between 195 and 196 of the CH1 domain, or between 388 and 389 of the CH3 domain of a parental antibody or fragment thereof.
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the peptide tag is grafted between amino acid residues 62 to 64 or 62 to 65 of the V H domain, or between amino acid residues 133 and 138 of the CH1 domain, or between 189 and 195 of the CH1 domain, or between 190 and 197 of the CH1 domain.
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the present invention provides modified antibodies or antibody fragments comprising SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, and/or SEQ ID NO:141.
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the present invention provides modified antibodies or antibody fragments comprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:139, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:
  • the present invention provides modified antibodies or antibody fragments comprising SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:374, SEQ ID NO:
  • the present invention provides modified antibodies or fragments thereof, which comprise at least one peptide tag that is a substrate of Sfp, and is located within the structural loop of said antibodies or antibody fragments, and wherein the peptide tag is GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWLVRCLN (SEQ ID NO:3), GDSLSWLLRCLN (SEQ ID NO:4), GDSLSWLVRLLN (SEQ ID NO:5), GDSLSWLLRSLN (SEQ ID NO:6), GSQDVLDSLEFIASKLA (SEQ ID NO:7), VLDSLEFIASKLA (SEQ ID NO:8), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), GDSLDMLEWSL (SEQ ID NO:11), GDSLDMLEWS (SEQ ID NO:12), GDSLDMLEW (SEQ ID NO:13), DSLDMLEW (SEQ ID NO:14),
  • the peptide tag is GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), DSLEFIASK (SEQ ID NO:19), or DSLDMLEWSL (SEQ ID NO: 1132).
  • the present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the modified antibodies or antibody fragments of the invention are an isotype selected from IgG, IgM, IgE and IgA. In some other embodiments, the modified antibodies or antibody fragments of the invention are a subtype of IgG selected from IgG1, IgG2, IgG3 and IgG4. In some embodiments, the modified antibodies or antibody fragments of the invention are a human or humanized antibody or antibody fragment. In a specific embodiment, the modified antibody or antibody fragment of the invention is an anti-HER2 antibody or anti-HER2 antibody fragment. The present invention further provides immunoconjugates comprising such modified antibodies or fragments thereof.
  • the present invention provides nucleic acids encoding the modified antibodies or antibody fragments described herein, and host cells comprising such nucleic acids.
  • the present invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein the modifice antibody or antibody fragment comprises at least one peptide tag that is a substrate of 4′-phosphopantetheinyl transferase, and is located within the structural loop of the antibody or antibody fragment.
  • the modified antibody or antibody fragment further comprises one or more orthogonal conjugation sites.
  • each orthogonal conjugation site is independently selected from a substrate of Sfp 4′-phosphopantetheinyl transferase, a substrate of AcpS 4′-phosphopantetheinyl transferase, a lysine, a cysteine, a tyrosine, a histidine, a formyl glycine, an unnatural amino acid, pyrrolysine and pyrroline-carboxylysine.
  • immunoconjugates comprising a modified antibody or antibody fragment, and a terminal group (TG) attached to the peptide tag in the modified antibody or antibody fragment by a linker having the structure according to Formula (I-b):
  • the terminal group is a drug moiety selected from an anti-inflammatory agent, an anticancer agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent and an anesthetic agent.
  • the drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an Eg5 inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, a proteasome inhibitor, a RNA polymerase inhibitor and a DHFR inhibitor.
  • the spectroscopic probe is selected from a fluorophore, a chromophore, a quantum dot, a magnetic probe, a radioactive probe, an imaging reagent, or a contrast reagent.
  • the affinity probe is biotin.
  • Another aspect provided herein is the preparation of an immunoconjugate by a process comprising the steps of:
  • Another aspect provided herein is the preparation of an immunoconjugate by a process comprising the steps of:
  • conjugated antibodies or antibody fragment thereof comprising the modified antibody or antibody fragment provided herein, wherein a serine residue of the peptide tag in the modified antibody or antibody fragment thereof is conjugated to a 4′-phosphopantetheine group having the structure of Formula (D-a), Formula (E-a), Formula (F-a) or Formula (G-a):
  • the conjugated serine has a structure selected from:
  • conjugated serine is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • conjugated antibodies or antibody fragment thereof comprising a modified antibody or antibody fragment thereof provided herein, wherein a serine residue of the peptide tag is conjugated to a modified 4′-phosphopantetheine group and the conjugated serine has a structure selected from:
  • the present invention also provides pharmaceutical compositions comprising an effective amount of the immunoconjugate of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient.
  • the present invention provides a method of treating a disease, such as cancer, comprising administering to a mammal in need thereof an effective amount of an immunoconjugate of the invention.
  • a disease such as cancer
  • the present invention provides immunoconjugates for use as a medicament.
  • the present invention provides use of an immunoconjugate in the manufacture of a medicament for treatment of cancer, autoimmune diseases, inflammatory diseases, infectious diseases (e.g., bacterial, fungus, virus), genetic disorders, cardiovascular diseases, and/or metabolic diseases.
  • the present invention provides methods of producing the immunoconjugates described herein.
  • the method comprises incubating a modified antibody or antibody fragment of the invention, a 4′-phosphopantetheinyl transferase, and a terminal group linked to CoA under suitable conditions to promote formation of an immunoconjugate comprising the antibody or antibody fragment and the terminal group linked together by 4′-phosphopantetheine.
  • the suitable condition comprises a temperature between 4° C. to 37° C. and pH 6.5 to pH 9.0.
  • the method comprising incubating under suitable conditions a modified antibody or antibody fragment of the invention, a 4′-phosphopantetheinyl transferase, and a terminal group linked to CoA or a terminal group linked to a CoA analogue, thereby promoting formation of the immunoconjugate which comprises the antibody or antibody fragment and the terminal group linked together by a linker comprising a 4′-phosphopantetheine or a 4′-phosphopantetheine analogue.
  • the suitable condition comprises a temperature between 4° C. to 37° C. and pH 6.5 to pH 9.0.
  • the method comprising comprises the steps:
  • the method comprising comprises the steps:
  • alkenyl or “alkene”, as used herein, refer to a branched or straight chain hydrocarbon having at least one carbon-carbon double bond. Atoms oriented about the double bond are in either the cis (Z) or trans (E) conformation.
  • C 2 -C 4 alkenyl refers to a branched or straight chain hydrocarbon having at least one carbon-carbon double bond and containing at least 2, and at most 4, 5, 6, 7 or 8 carbon atoms, respectively.
  • alkenyl groups include ethenyl, ethane, epropenyl, propene, allyl (2-propenyl), 2-propene, butenyl, pentenyl, pentene, hexenyl, heptenyl, heptene, octenyl, nonenyl, nonene, decenyl, decene and the like. If not otherwise specified, an alkenyl group generally is a C 2 -C 6 alkenyl.
  • alkynyl or “alkyne”, as used herein, refer to a branched or straight chain hydrocarbon radical having at least one carbon-carbon triple bond.
  • C 2 -C 4 alkynyl refers to a branched or straight chain hydrocarbon radical having at least one carbon-carbon triple bond and containing at least 2, and at most 4, 5, 6, 7 or 8 carbon atoms, respectively.
  • alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl and the like. If not otherwise specified, an alkynyl group generally is a C 2 -C 6 alkynyl.
  • alkyl refers to a saturated branched or straight chain hydrocarbon.
  • C 1 -C 3 alkyl refers to saturated branched or straight chain hydrocarbon.
  • C 1 -C 4 alkyl refers to saturated branched or straight chain hydrocarbon containing at least 1, and at most 3, 4, 5, 6, 7 or 8 carbon atoms, respectively.
  • alkyl groups as used herein include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl and the like. If not otherwise specified, an alkyl group generally is a C 1 -C 6 alkyl.
  • alkoxy refers to the group —OR a , where R a is an alkyl group as defined herein.
  • R a is an alkyl group as defined herein.
  • C 1 -C 3 alkoxy refers to an alkoxy group wherein the alkyl moiety contains at least 1, and at most 3, 4, 5, 6, 7 or 8, carbon atoms.
  • Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy, t-butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy and the like.
  • aryl refers to monocyclic, bicyclic, and tricyclic ring systems having a total of six to fourteen ring members, wherein at least one ring in the system is aromatic.
  • An aryl group also includes one or more aromatic rings fused to one or more non-aromatic hydrocarbon rings.
  • Non-limiting examples of aryl groups, as used herein, include phenyl (Ph), naphthyl, fluorenyl, indenyl, azulenyl, anthracenyl and the like.
  • An aryl group may contain one or more substituents and thus may be “optionally substituted”. Unless otherwise specified, aryl groups can have up to four substituents.
  • cycloalkyl refers to a saturated monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring assembly.
  • C 3 -C 5 cycloalkyl refers to a saturated monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring assembly which contains at least 3, and at most 5, 6, 7, 8, 9 or 10, carbon atoms.
  • Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, decahydronaphthalenyl and the like. If not otherwise specified, a cycloalkyl group generally is a C 3 -C 8 cycloalkyl.
  • cycloalkenyl or “cycloalkene”, as used herein, refers to a monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring assembly having at least one carbon-carbon double bond. Atoms oriented about the double bond are in either the cis (Z) or trans (E) conformation. A monocyclic cycloalkene can be fused to one or two aryl rings.
  • Non-limiting examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, cyclodecenyl, and the like. If not otherwise specified, a cycloalkenyl group generally is a C 5 -C 8 cycloalkenyl.
  • cycloalkynyl refers to a monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring assembly having at least one carbon-carbon triple bond.
  • a monocyclic cycloalkyne can be fused to one or two aryl rings.
  • Non-limiting examples of cycloalkynyl groups, as used herein, include cyclopropynyl, cyclobutynyl, cyclopentynyl, cyclohexynyl, cycloheptynyl, cyclooctynyl, cyclononynyl, cyclodecynyl, and the like. If not otherwise specified, a cycloalkynyl group generally is a C 6 -C 8 cycloalkynyl.
  • heteroaryl refers to a 5-6 membered heteroaromatic monocyclic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, an 8-10 membered fused bicyclic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur as ring members and where at least one of the rings is aromatic, or a 12-14 membered fused tricyclic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur and where at least one of the rings is aromatic.
  • fused bicyclic and tricyclic ring systems may be fused to one or more aryl, cycloalkyl, or heterocycloalkyl rings.
  • heteroaryl groups include 2- or 3-furyl; 1-, 2-, 4-, or 5-imidazolyl; 3-, 4-, or 5-isothiazolyl; 3-, 4-, or 5-isoxazolyl; 2-, 4-, or 5-oxazolyl; 4- or 5-1,2,3-oxadiazolyl; 2- or 3-pyrazinyl; 1-, 3-, 4-, or 5-pyrazolyl; 3-, 4-, 5- or 6-pyridazinyl; 2-, 3-, or 4-pyridyl; 2-, 4-, 5- or 6-pyrimidinyl; 1-, 2- or 3-pyrrolyl; 1- or 5-tetrazolyl; 2- or 5-1,3,4-thiadiazolyl; 2-, 4-, or 5-thiazolyl; 2- or 3-thienyl; 2-, 4- or 6-1,3,5-triazinyl; 1-, 3- or 5-1,2,4-triazolyl; 1-, 4- or 5-1,2,3-triazolyl; 1-, 2-,
  • heteroatoms refers to nitrogen (N), oxygen (O) or sulfur (S) atoms.
  • heterocycloalkyl refers to a to saturated 3-8 membered monocyclic hydrocarbon ring structure, a saturated 6-9 membered fused bicyclic hydrocarbon ring structure, or a saturated 10-14 membered fused tricyclic hydrocarbon ring structure, wherein one to four of the ring carbons of the hydrocarbon ring structure are replaced by one to four groups independently selected from —O—, —NR—, and —S—, wherein R is hydrogen, C 1 -C 4 alkyl or an amino protecting group.
  • heterocycloalkyl groups include aziridinyl, aziridin-1-yl, aziridin-2-yl, aziridin-3-yl, oxiranyl, oxiran-2-yl, oxiran-3-yl, thiiranyl, thiiran-2-yl, thiiran-3-yl, azetadinyl, azetadin-1-yl, azetadin-2-yl, azetadin-3-yl, oxetanyl, oxetan-2-yl, oxetan-3-yl, oxetan-4-yl, thietanyl, thietan-2-yl, thietan-3-yl, thietan-4-yl, pyrrolidinyl, pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, thietan-4-yl,
  • the term “optionally substituted”, as used herein, means that the referenced group may or may not be substituted with one or more additional group(s) in place of one or more hydrogen atoms of the unsubstituted group.
  • the number of such groups that can be present ranges from one up to the number of hydrogen atoms on the unsubstituted group.
  • the optional substituents are individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof.
  • Non-limiting examples of optional substituents include, halo (particularly F, Cl and Br), —CN, —OR, —R, —NO 2 , —C( ⁇ O)R, —OC( ⁇ O)R, —C( ⁇ O)OR, —OC( ⁇ O)NHR, —C( ⁇ O)N(R) 2 , —SR—, —S( ⁇ O)R, —S( ⁇ O) 2 R, —NHR, —N(R) 2 , —NHC( ⁇ O)R, —NRC( ⁇ O)R, —NRC(S)R, NHC( ⁇ O)OR, —NRCO 2 R, —NRC( ⁇ O)N(R) 2 , —NRC(S)N(R) 2 , —NRNRC( ⁇ O)R, —NRNRC( ⁇ O)N(R) 2 , —NRNRCO 2 R, —C( ⁇ O)NH—, S( ⁇ O) 2 N
  • Suitable substituents for alkyl, cycloalkyl, and heterocycloalkyl groups can further include ⁇ CHR, ⁇ O (oxo) and ⁇ N—R.
  • Preferred substituents for an aryl or heteroaryl group are selected from F, Cl, Br, CN, —NR′ 2 , hydroxy, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy, C 1 -C 4 alkoxy-C 1 -C 4 alkyl, —COOR′, —CONR′ 2 , —SR′, and —SO 2 R′, where each R′ is H or C 1 -C 4 alkyl.
  • Preferred substituents for an alkyl, cycloalkyl or heterocycloalkyl group are selected from oxo ( ⁇ O), F, Cl, Br, CN, —NR′ 2 , hydroxy, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy, C 1 -C 4 alkoxy-C 1 -C 4 alkyl, —COOR′, —CONR′ 2 , —SR′, and —SO 2 R′, where each R′ is H or C 1 -C 4 alkyl.
  • amino acid refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • unnatural amino acid is intended to represent amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism, whether the same or different.
  • unnatural amino acids require a modified tRNA and a modified tRNA synthetase (RS) for incorporation into a protein.
  • RS modified tRNA synthetase
  • These “selected” orthogonal tRNA/RS pair are specific for the unnatural amino acid and are generated by a selection process as developed by Schultz et al. (see, e.g., Liu et al., Annu. Rev. Biochem. 79:413-444, 2010) or a similar procedure.
  • unnatural amino acid does not include the natural occurring 22 nd proteinogenic amino acid pyrrolysine (Pyl) as well as its demethylated analogue pyrroline-carboxy-lysine (Pcl), because incorporation of both residues into proteins is mediated by the unmodified, naturally occurring pyrrolysyl-tRNA/tRNA synthetase pair (see, e.g., Ou et al., Proc. Natl. Acad. Sci. USA. 108:10437-10442, 2011).
  • antibody refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner.
  • a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention).
  • the antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).
  • variable domains of both the light (V L ) and heavy (V H ) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (C L ) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody.
  • the N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and C L domains actually comprise the carboxy-terminal domains of the heavy and light chain, respectively.
  • antibody fragment refers to either an antigen binding fragment of an antibody or a non-antigen binding fragment (e.g., Fc) of an antibody.
  • antigen binding fragment refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab′) fragments, a monovalent fragment consisting of the V L , V H , C L and CH1 domains; a F(ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V H and CH1 domains; a Fv fragment consisting of the V L and V H domains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a V H domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.
  • scFv single-chain Fvs
  • sdFv disulfide-linked Fvs
  • Fab fragments F(
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (“scFv”); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen binding fragment.” These antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).
  • Fn3 fibronectin type III
  • Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (V H -CH1-V H -CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Pat. No. 5,641,870).
  • monoclonal antibody or “monoclonal antibody composition” as used herein refers to polypeptides, including antibodies and antibody fragments that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human antibody includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000).
  • the human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).
  • humanized antibody refers to an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).
  • epitopes refers to an antibody or antigen binding fragment thereof that finds and interacts (e.g., binds) with its epitope, whether that epitope is linear or conformational.
  • epitope refers to a site on an antigen to which an antibody or antigen binding fragment of the invention specifically binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
  • affinity refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • conservatively modified variant refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • “conservatively modified variants” include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term “conservative sequence modifications” are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • the term “optimized” as used herein refers to a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell.
  • the optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the “parental” sequence.
  • percent identical in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).
  • operably linked in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • immunoconjugate or “antibody conjugate” as used herein refers to the linkage of an antibody or an antibody fragment thereof with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, and the like.
  • the linkage can be covalent bonds, or non-covalent interactions such as through electrostatic forces.
  • Various linkers known in the art, can be employed in order to form the immunoconjugate.
  • the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate.
  • fusion protein refers to proteins created through the joining of two or more genes or gene fragments which originally coded for separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • cytotoxin refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.
  • anti-cancer agent refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.
  • terminal group refers to a chemical moiety or a surface that is conjugated to the antibody or antibody fragment of the invention.
  • a terminal group can be a drug moiety selected from an anti-cancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, an anesthetic agent.
  • a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a RNA polymerase inhibitor, an Eg5 inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • a MetAP methionine aminopeptidase
  • an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor an inhibitors of
  • Suitable examples include auristatins such as MMAE and MMAF; calicheamycins such as gamma-calicheamycin; and maytansinoids such as DM1 and DM4.
  • auristatins such as MMAE and MMAF
  • calicheamycins such as gamma-calicheamycin
  • maytansinoids such as DM1 and DM4.
  • a terminal group can be a biophysical probe, a fluorophore, a spin label, an infrared probe an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, or a polysaccharide.
  • such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • Tumor refers to neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • anti-tumor activity means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity.
  • a possible way of showing anti-tumor activity is to show a decline in growth rate of abnormal cells that arises during therapy or tumor size stability or reduction.
  • Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer includes a malignancy characterized by deregulated or uncontrolled cell growth.
  • Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas.
  • cancer includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).
  • primary malignant tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor
  • secondary malignant tumors e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • insertion in the context of inserting a peptide tag into an antibody means the incorporation of a peptide tag between two specific residues of an antibody. The total number of residues of the antibody is increased by the number of inserted tag residues.
  • grafting in the context of incorporating a peptide tag into an antibody refers to the incorporation of a peptide tag into an antibody by mutagenesis. For instance, a short stretch of amino acid residues within a non-CDR loop is substituted by a peptide sequence. In this case, the total number of residues of the antibody remains unchanged.
  • the term “grafting” also encompasses a combination of substitution and insertion of peptide tag residues. For example, one part of the peptide tag is incorporated by substitution of structural loop residues, while the remaining part is inserted between specific residues of the non-CDR loop. The total number of residues of the IgG antibody is increased by a number that is smaller than the number of tag residues,
  • FIG. 1 Schematic description of 4′-phosphopantetheinyl transferase (PPTase)-mediated generation of ADCs.
  • FIG. 2 Design of IgG1 constructs which contain peptide tags for site-specific antibody labeling via post-translational 4′-phosphopantetheinylation.
  • IgG1 constructs contain peptide tags (underlined) in the V H , CH1, and CH3 domains.
  • IgG1 constructs contain peptide tags (underlined) in the CH3, V L , and C L domains.
  • Designed constructs that were successfully cloned are marked by a plus (+) sign in the left column. Unsuccessful cloning is indicated by a minus ( ⁇ ) sign.
  • Successfully cloned constructs are grouped as non-expressors ( ⁇ ) and expressors (+) (middle column).
  • FIG. 2(A) discloses residues 1-68 of SEQ ID NO: 1130, residues 1-80 of SEQ ID NO: 94, residues 1-79 of SEQ ID NO: 95, residues 1-80 of SEQ ID NO: 96, residues 1-72 of SEQ ID NO: 1130, residues 1-80 of SEQ ID NO: 99, residues 1-79 of SEQ ID NO: 97, residues 1-77 of SEQ ID NO: 98, residues 122-198 of SEQ ID NO: 1130, residues 1-77 of SEQ ID NO: 100, residues 1-77 of SEQ ID NO: 102, residues 1-77 of SEQ ID NO: 101, residues 1-77 of SEQ ID NO: 105, residues 1-77 of SEQ ID NO: 107, residues 122-190 of SEQ ID NO: 1130, residues 1-76 of SEQ ID NO: 108, residues 1-75 of SEQ ID NO: 103, residues 1-74 of SEQ ID NO: 106, residues 164-231 of SEQ ID NO: 11
  • FIG. 2(B) discloses residues 324-388 of SEQ ID NO: 1130, residues 203-278 of SEQ ID NO: 122, residues 203-279 of SEQ ID NO: 121, residues 373-449 of SEQ ID NO: 1130, residues 252-328 of SEQ ID NO: 124, residues 252-328 of SEQ ID NO: 125, residues 252-328 of SEQ ID NO: 135, residues 252-328 of SEQ ID NO: 137, residues 252-328 of SEQ ID NO: 138, residues 373-444 of SEQ ID NO: 1130, residues 252-328 of SEQ ID NO: 134, residues 390-449 of SEQ ID NO: 1130, residues 269-340 of SEQ ID NO: 127, residues 269-335 of SEQ ID NO: 126, residues 269-339 of SEQ ID NO: 129, residues 269-337 of SEQ ID NO: 131, residues 269-338 of SEQ ID NO: 130, residue
  • FIG. 3 (A) Sequence of CH1 domain, CH2 domain, CH3 domain, and hinge region of the Ig gamma 1 heavy chain (SEQ ID NO:93). (B) Sequence of C L domain of the Ig kappa light chain (SEQ ID NO:24). Underlined amino acids are structural loops. Amino acid positions are numbered according to the Eu numbering system as described in Edelman et al., Proc. Natl. Acad. USA 63:78-85 (1969).
  • X′ 1 , X′ 2 , X′ 3 , X′ 4 , X′ 5 , and X′ 6 indicate residues that are present at allotypic positions within the IgG1 subclass and the kappa isotype (according to Jefferis et al., MAbs. 1:332-338 (2009)).
  • FIG. 4 (A) Sequence alignment of CH1 domain, CH2 domain, CH3 domain, and hinge region of the four human Ig gamma subclasses with Trastuzumab (SEQ ID NOs 1109-1113, respectively, in order of appearance). (B) Sequence alignment of C L domain with Trastuzumab (SEQ ID NOs 1114-1115, respectively, in order of appearance). Underlined residues belong to structural loops (see also FIG. 3 ). Boxed residues indicate allotypic positions according to Jefferis et al., MAbs. 1:332-338 (2009). For simplicity, only the allotypic positions within the IgG1 subclass and the kappa isotype are shown. Protein sequences of the human Ig gamma subclasses and the human kappa isotype are derived from the UniProt database (entry numbers P01857, P01859, P01860, P01861, and P01834).
  • FIG. 5 HPLC characterization of Sfp-catalyzed ADC formation.
  • A HPLC trace confirming the near quantitative formation of the immunoconjugate anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360 (SEQ ID NO:1117).
  • B HPLC trace confirming the near quantitative formation of the immunoconjugate anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 (SEQ ID NO:1118).
  • C HPLC trace confirming the near quantitative formation of the immunoconjugate anti-hHER2-HC-V2-DS-ppan-MC-MMAF-LEFIASKLA-Q3 (SEQ ID NO:1119).
  • FIG. 6 Characterization of three trastuzumab immunoconjugates by analytical size-exclusion chromatography (AnSEC) exemplifies the formation of monomeric, non-aggregated ADCs.
  • A AnSEC analysis of the immunoconjugate anti-hHER2-HC-V2-GDS-ppan-MC-MMAF-LSWLLRLLN-Q3 (SEQ ID NO:1120).
  • B AnSEC analysis of the immunoconjugate anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389 (SEQ ID NO:1122).
  • C AnSEC analysis of the immunoconjugate anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKL-N389 (SEQ ID NO:1121).
  • FIG. 7 HPLC characterization of unsuccessful labeling of trastuzumab with incorporation of a peptide tag at a specific location.
  • HPLC trace indicating no conjugation between anti-hHER2-HC-S190D-S191-S192L-L193E-G194F-T195I-Q196A-T197S-Y198K-I199L (SEQ ID NO:114) and CoA-MC-MMAF.
  • FIG. 8 HPLC characterization of the labeling of mixed grafting/insertion constructs with CoA-MC-MMAF.
  • A HPLC trace indicating partial formation of the immunoconjugate anti-hHER2-HC-S63-ppan-MC-MMAF-V64L-EFIASKLA-K65 (SEQ ID NO:1125).
  • B HPLC trace indicating no formation of the immunoconjugate anti-hHER2-LC-S76D-S77-ppan-MC-MMAF-L78-EFIASKLA-Q79 (SEQ ID NO:1126).
  • FIG. 9 HPLC characterization of fluorophore attachment to IgGs.
  • A HPLC trace confirming the near quantitative formation of the antibody-fluorophore conjugate anti-hHER2-HC-P189G-S190D-S191-ppan-maleimidoethylamido-TMR-S192L-L193S-G194W-T195L (SEQ ID NO:1127). The extensive overlap between the HPLC traces monitored at 280 and 555 nm indicates near quantitative fluorophore conjugation.
  • FIG. 10 HPLC characterization of antibody labeling with hydrolyzed maleimido- or bromoacetyl thioether-linked cytotoxins.
  • A HPLC trace confirming the near quantitative conjugation of maleimide-ring-opened CoA-MC-MMAF to anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:121).
  • B HPLC trace confirming the near quantitative conjugation of CoA-Ac-Ahx-MMAF to anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:121).
  • FIG. 11 HPLC characterization of antibody labeling with cytotoxins connected via a cleavable linker.
  • A HPLC trace confirming the near quantitative conjugation of CoA-MC-Val-Cit-PABC-MMAF to anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:121).
  • B HPLC trace confirming the near quantitative conjugation of CoA-MC-Val-Cit-PABC-MMAF to anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ ID NO:127).
  • FIG. 12 Optimization of 4′-phosphopantetheinyl transferase (PPTase)-catalyzed ADC formation as a function of pH.
  • the bar graph representation shows the amount of generated ADC with a drug-to-antibody ratio (DAR) of 2 as a function of pH.
  • DAR drug-to-antibody ratio
  • the data is based on the HPLC analysis (280 nm) of the reaction of CoA-MC-MMAF with either anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:121) or anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ ID NO:127) at a pH range of 5.0 to 10.0.
  • FIG. 13 Optimization of conjugation reaction as a function of Sfp enzyme concentration in 50 mM HEPES buffer (pH 7.5) containing 2.5 ⁇ M antibody, 50 ⁇ M CoA-MC-MMAF, and 10 mM MgCl 2 (37° C., 16 hours).
  • A Deconvoluted mass spectrum showing primarily unconjugated anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ ID NO:127) at an Sfp concentration of 0.1 ⁇ M.
  • FIG. 14 Optimization of enzymatic conjugation reaction as a function of CoA-MC-MMAF substrate concentration at pH 8.0.
  • the HPLC traces represent three conjugation reactions with 2.5 ⁇ M anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ ID NO:127) that contained 2.5 ⁇ M (top trace), 7.5 ⁇ M (middle trace), or 25 ⁇ M (bottom trace) of CoA-MC-MMAF.
  • the bar graph representation shows the amount of generated ADC with a DAR of 2 as a function of CoA-MC-MMAF substrate concentration.
  • the titration series was performed at an Sfp enzyme concentration of either 0.25 ⁇ M (black bars) or 1.0 ⁇ M (white bars).
  • FIG. 15 Thermal stability of peptide-tagged ADCs as measured by differential scanning fluorometry (DSF) using SYPRO Orange gel stain.
  • DSF differential scanning fluorometry
  • FIG. 16 Pharmacokinetic (PK) study of two peptide-tagged Trastuzumab immunoconjugates. Plasma titers of both ADCs were determined by capturing the respective immunoconjugates with plate-absorbed human HER2 (extracellular domains 3-4) followed by detection with anti-human IgG and anti-MMAF antibodies.
  • PK Pharmacokinetic
  • FIG. 17 In vitro cell-killing assay of peptide-tagged immunoconjugates using the HER2-expressing MDA-231 cell line. Plots are based on cell viability measurements using the Cell Titer Glo Luminescent Cell Viability Assay (Promega).
  • Figure discloses ‘anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360,’ ‘anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389,’ ‘anti-hHER2-HC-T359-GDS-ppan-MC-ValCit-PABC-MMAF-LSWLLRLLN-K360,’ and ‘anti-hHER2-HC-E388-GDS-ppan-MC-ValCit-PABC-MMAF-LSWLLRLLN-N389’ as SEQ ID NOS 1117, 1118, 1108, and 1107, respectively.
  • FIG. 18 Plot illustrating the influence of peptide tag insertion site on IgG antibody thermal stability.
  • Tm1 first transition temperature
  • DSF differential scanning fluorometry
  • peptide tag insertions in the CH1 domain of the Fab region destabilize the antibody scaffold to a much lesser extent, with Tm1 values generally not more than 3 degree Celcius lower than unmodified Trastuzumab IgG1 with a Tm1 of 69.7° C.
  • FIG. 19 Enzymatic generation of ADCs with a DAR of 4.
  • ADCs with a DAR of 4 can be generated by incorporating multiple peptide tags into an antibody, such as the ybbR— and the S6-tags.
  • B HPLC analysis of Sfp-catalyzed conjugation of CoA-MC-MMAF to Trastuzumab IgG containing an S6 tag in the V H domain as well as a ybbR tag in the CH3 domain (anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388-DSLEFIASKLA-N389 (SEQ ID NO:142)).
  • FIG. 20 Pharmacokinetic profiles of peptide-tagged trastuzumab immunoconjugates displaying high and low AUC IgG values.
  • Each of the six peptide-tagged ADCs corresponding to SEQ ID NO:248 (A), SEQ ID NO:33 (B), SEQ ID NO:251 (C), SEQ ID NO:218 (D), SEQ ID NO:202 (E), and SEQ ID NO:244 (F) was administered intravenously into three mice at a single dose of 1 mg/kg. After collection of plasma samples over a time period of 340 hours, trastuzumab ADC molecules were captured by using the immobilized extracellular domain of human HER2.
  • Plasma titers were then determined by two ELISA formats based on either anti-MMAF or anti-hIgG antibodies. While the first format provides readout on the concentration of “intact” ADC, the latter format generates a signal proportional to the concentration of total IgG, comprising both conjugated and unconjugated trastuzumab molecules.
  • a C exemplify PK curves of peptide-tagged MMAF ADCs displaying high AUC IgG values, whereas D F show examples of immunoconjugates exhibiting very low AUC IgG values. In all cases, anti-MMAF and anti-hIgG titers closely parallel each other indicating negligible deconjugation of the MMAF payload during the time course of the PK study.
  • FIG. 21 Correlation between anti-MMAF and anti-hIgG titers of 86 peptide-tagged ADCs. According to this plot, the concentration readouts of total IgG and “intact” ADC are in close agreement to each other, thereby suggesting a highly stable ppan-MC linkage between MMAF payload and peptide-tagged antibody. Besides negligible deconjugation of the MMAF drug in vivo, this highly linear correlation also indicates that covalent payload attachment does not negatively affect the pharmacokinetic profile of the immunoconjugate.
  • FIG. 22 Two-step method involving the post-translational modification of an A1-tagged antibody with a carbonyl-functionalized CoA analogue for subsequent attachment of the terminal group (TG) via oxime ligation.
  • the A1-tagged antibody is site-specifically labeled with a ketone- or aldehyde-functionalized CoA analogue in cell-culture medium.
  • the carbonyl group of the ppan moiety is reacted with an aminooxy-derivatized TG.
  • FIG. 23 In vivo efficacy study of the ybbR-tagged trastuzumab ADC anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389 (SEQ ID NO: 1122) in immune-deficient nude mice implanted with a human tumor cell line.
  • the xenograft tumor model was performed with nu/nu mice which were subcutaneously administered with the HER2-dependent breast cancer cell line MDA-MB231 clone 16.
  • the present invention provides methods of site-specific labeling of antibodies, using proteins having 4′-phosphopantetheinyl transferase activity (“PPTases”) that catalyze post-translational modification of peptide sequences (“peptide tags”) incorporated into one or more specific sites of an antibody of interest.
  • PPTases proteins having 4′-phosphopantetheinyl transferase activity
  • peptide tags peptide sequences incorporated into one or more specific sites of an antibody of interest.
  • Enzymatic labeling under ambient reaction conditions enables quantitative and irreversible covalent modification of a specific serine residue within the peptide tags incorporated into the antibody, and thus creates desirable antibody conjugates.
  • site-specific antibody labeling can be achieved with a variety of chemically accessible labeling reagents, such as anti-cancer agents, fluorophores, peptides, sugars, detergents, polyethylene glycols, immune potentiators, radio-imaging probes, prodrugs, and other molecules.
  • PPTases can be used to immobilize peptide-tagged antibodies on solid support, such as polystyrene nanoparticles and gold surfaces (see, e.g., Wong et al., Org. Biomol. Chem. 8: 782-787, 2010; Wong et al., Nanoscale 4:659-666, 2012, for methodology of immobilization of functional enzymes).
  • the present invention provides methods of preparation of homogeneous immunoconjugates with a defined drug-to-antibody ratio for use in cancer therapy, and immunoconjugates prepared thereby, as well as pharmaceutical compositions comprising these immunoconjugates.
  • the methods of the instant invention can be used in combination with other conjugation methods known in the art.
  • a “structural loop” or “non-CDR-loop” is to be understood in the following manner: antibodies are made of domains with immunoglobulin folds. In essence, anti-parallel beta sheets are connected by loops to form a compressed antiparallel beta barrel. In the variable region, some of the loops of the domains contribute essentially to the specificity of the antibody, i.e., the binding to an antigen. These loops are called “CDR-loops.” All other loops of antibody domains are rather contributing to the structure of the molecule and/or the effector function. These loops are defined herein as “structural loops” or “non-CDR-loops.”
  • the antibodies (e.g., a parent or native antibody, optionally containing one or more non-naturally occurring amino acids) of the present invention are numbered according to the Eu numbering system as set forth in Edelman et al., Proc. Natl. Acad. USA 63:78-85 (1969).
  • Human IgG1 constant region is used as a representative throughout the application.
  • the invention is not limited to human IgG1; corresponding amino acid positions can be readily deduced by sequence alignment.
  • FIG. 3 (A) shows IgG1 heavy chain constant region where the structural loops are underlined, these underlined structural loops can be readily identified for IgG2, IgG3, and IgG4 as shown in the sequence alignment of FIG. 4 (A).
  • FIG. 3 (A) shows IgG1 heavy chain constant region where the structural loops are underlined, these underlined structural loops can be readily identified for IgG2, IgG3, and IgG4 as shown in the sequence alignment of FIG. 4 (A).
  • FIG. 3 shows the light chain constant region where the structural loops are underlined.
  • IgG1, IgG2, IgG3 and IgG4 are the same.
  • Table 1 below lists the amino acid positions in the structural loop of IgG1, IgG2, IgG3 and IgG4, respectively.
  • FIG. 3 as well as SEQ ID NOs 24 and 93 represent the sequences of the Ig kappa light chain constant region and the Ig gamma-1 heavy chain constant region, respectively.
  • X′ 1 , X′ 2 , X′ 3 , X′ 4 , X′ 5 , and X′ 6 in SEQ ID NOs: 24 and 93 indicate residues that are present at allotypic positions within the IgG1 subclass and the kappa isotype (according to Jefferis et al., MAbs. 1:332-338 (2009)).
  • X′ 1 can be Arg or Lys
  • X′ 2 can be Asp or Glu
  • X′ 3 can be Leu or Met
  • X′ 4 can be Ala or Gly
  • X′ 5 can be Val or Ala
  • X′ 6 can be Leu or Val.
  • findings of the invention are not limited to any specific antibodies.
  • the findings of the invention are not limited to using PPTases.
  • the positions in the antibody structural loops identified herein can also be used for incorporating other peptide tags, which are substrates for other enzymatic conjugation approaches such as the enzyme biotin protein ligase (BPL), transglutaminases, and formylglycine forming enzymes.
  • BPL biotin protein ligase
  • transglutaminases transglutaminases
  • formylglycine forming enzymes formylglycine forming enzymes.
  • the present invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or fragment thereof comprises a peptide tag that by itself is a substrate of a 4′-phosphopantetheinyl transferase, and wherein said peptide tag is located within a structural loop, or C- or N-terminus of the modified antibody or fragment thereof.
  • the present invention also provides modified antibodies or fragments thereof comprising a peptide tag that is a substrate of a 4′-phosphopantetheinyl transferase, and wherein said peptide tag is located within a structural loop, or C- or N-terminus of the antibody or fragment thereof.
  • said peptide tag is one or more peptides selected from those described in Table 2.
  • the peptide tag is inserted between two amino acids of a structural loop of said antibody or fragment thereof.
  • the peptide tag is grafted into a structural loop, C- or N-terminus of said antibody or fragment thereof, wherein the peptide tag replaces one or more amino acids of the parent antibody or fragment thereof.
  • the structural loop refers to a structural loop located at the CH1, CH2, CH3, or C L region of said antibody or fragment thereof.
  • the modified antibody heavy chain and/or light chain (or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its structural loops.
  • the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more protein tags in its structural loops.
  • said 4′-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a mutant form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • said 4′-phosphopantetheinyl transferase originates from Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima, Clostridium thermocellum , as well as any other mammalian, bacterial or fungal genome.
  • said 4′-phosphopantetheinyl transferase is a homologous protein to Sfp, AcpS, T. maritima PPTase, human PPTase, or a mutant thereof. In one embodiment, said 4′-phosphopantetheinyl transferase is from a thermophilic organism.
  • the parental antibody (antibody without incorporating the peptide tag) is an IgG, IgM, IgE, or IgA antibody. In some embodiments, the parental antibody is an IgG1 antibody. In some other embodiments, the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • a substrate of 4′-phosphopantetheinyl transferase as used herein means the structure being described can serve as an acceptor for a 4′-phosphopantetheine (ppan) or modified ppan group as illustrated in Scheme 1a herein when contacted with 4′-phosphopantetheinyl transferase and CoA or a CoA analogue having a terminal group attached to it.
  • the present invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or fragment thereof comprises a CH1, CH2, CH3, and/or C L region, and wherein said CH1, CH2, CH3, and/or C L region further comprises a peptide tag that by itself is a substrate of a 4′-phosphopantetheinyl transferase.
  • the present invention also provides modified antibodies or fragments thereof comprising a CH1, CH2, CH3, and/or C L region, and wherein said CH1, CH2, CH3, and/or C L region further comprises a peptide tag that is a substrate of a 4′-phosphopantetheinyl transferase.
  • said peptide tag is one or more peptides selected from those described in Table 2. In some embodiments, the peptide tag is inserted between two amino acids of a structural loop of said antibody or fragment thereof. In some embodiments, the peptide tag is grafted into a structural loop of said antibody or fragment thereof.
  • the modified antibody heavy chain and/or light chain (or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its structural loops. In some embodiments, the modified antibodies or fragments contain 2, 4, 6, 8, or more protein tags in its structural loops. In some embodiments, said 4′-phosphopantetheinyl transferase is Sfp, AcpS, T.
  • maritima PPTase human PPTase, or a mutant form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • said 4′-phosphopantetheinyl transferase originates from Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima, Clostridium thermocellum , as well as any other mammalian, bacterial or fungal genome.
  • said 4′-phosphopantetheinyl transferase is a homologous protein to Sfp, AcpS, T. maritima PPTase, or a mutant thereof.
  • said 4′-phosphopantetheinyl transferase is from a thermophilic organism.
  • the parental antibody is an IgG, IgM, IgE, or IgA antibody.
  • the parental antibody is an IgG1 antibody.
  • the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • “retains” activity means the enzyme being described maintains at least about 10% of the activity of the reference material, which is the B. subtilis Sfp 4′-phosphopantetheinyl transferase (see, e.g., Quadri et al., Biochemistry 37: 1585-1595 (1998)).
  • the reference material which is the B. subtilis Sfp 4′-phosphopantetheinyl transferase (see, e.g., Quadri et al., Biochemistry 37: 1585-1595 (1998)).
  • a different 4′-phosphopantetheinyl transferase or a mutant form of the enzyme retains at least about 10% of the 4′-phosphopantetheinyl transferase activity compared to Sfp under identical reaction conditions, i.e., using the same CoA substrate, the same peptide-tagged antibody, identical buffer conditions, identical substrate and enzyme concentrations, the same temperature, and the same reaction duration.
  • the present invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or fragment thereof comprises a peptide tag that by itself is a substrate of a 4′-phosphopantetheinyl transferase, and wherein said peptide tag is inserted between positions 2 and 3 of the V H domain, positions 63 and 64 of the V H domain, positions 64 and 65 of the V H domain, positions 138 and 139 of the CH1 domain, positions 197 and 198 of the CH1 domain, positions 359 and 360 of the CH3 domain, positions 388 and 389 of the CH3 domain, the C-terminus of the CH3 domain (after Lys447), and/or positions 2 and 3 of the V L domain of a parental antibody or fragment thereof.
  • the present invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or fragment thereof comprises a peptide tag that by itself is a substrate of a 4′-phosphopantetheinyl transferase, and wherein the peptide tag is inserted between amino acid residues 2 and 3 of the VH or VL domain, or between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 164 and 165, or between 165 and 166, or between 194 and 195 of the CH1 domain, or between 388 and 389, or between 445 and 446, or between 446 and 447 of the CH3 domain of a parental antibody or fragment thereof.
  • the peptide tag is inserted between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 165 and 166 of the CH1 domain, or between 388 and 389 of the CH3 domain of a parental antibody or fragment thereof,
  • the invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or fragment thereof comprises SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, and/or SEQ ID NO:141.
  • the invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or antibody fragment comprises comprises SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:139, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:26,
  • the modified antibody or antibody fragment comprises SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:169, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:268, SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:374, or SEQ ID NO:384.
  • said peptide tag is one or more peptides selected from those described in Table 2.
  • the modified antibody heavy chain and/or light chain (or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its structural loops.
  • the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more protein tags in its structural loops.
  • said 4′-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a mutant form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • said 4′-phosphopantetheinyl transferase originates from Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima, Clostridium thermocellum , as well as any other mammalian, bacterial or fungal genome.
  • said 4′-phosphopantetheinyl transferase is Sfp and the peptide tag is selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19).
  • the parental antibody is an IgG, IgM, IgE, or IgA antibody.
  • the parental antibody is an IgG1 antibody.
  • the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • the present invention provides immunoconjugates comprising a modified antibody or fragment thereof, and a terminal group, wherein said modified antibody or fragment thereof comprises a peptide tag that by itself is a substrate of a 4′-phosphopantetheinyl transferase, and wherein said peptide tag is grafted into a structural loop, or C- or N-terminus of the antibody or fragment thereof.
  • said peptide tag is grafted at amino acid positions from 62 to 64 of the V H domain (mutations at amino acids 62 and 63, and insertion of the rest of the peptide tag between amino acids 63 and 64), at amino acid positions from 62 to 65 of the V H domain (mutations at amino acids 62-64, and insertion of the rest of the peptide tag between amino acids 64 and 65); at amino acid positions from 133 to 139 of the CH1 domain (mutations of amino acids 133-138, and insertion of the rest of the peptide tag between amino acids 138-139), amino acid positions from 189 to 195 of the CH1 domain, and/or amino acid positions from 190 to 198 of the CH1 domain (mutations from amino acids 190-197, and insertion of the rest of the peptide tag between 197 and 198) of a parental antibody or fragment thereof.
  • said peptide tag is one or more peptides selected from those described in Table 2.
  • the modified antibody heavy chain and/or light chain (or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its structural loops.
  • the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more protein tags in its structural loops.
  • said 4′-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a mutant form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • said 4′-phosphopantetheinyl transferase originates from Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima, Clostridium thermocellum , as well as any other mammalian, bacterial or fungal genome.
  • said 4′-phosphopantetheinyl transferase is Sfp and the peptide tag is selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19).
  • the parental antibody is an IgG, IgM, IgE, or IgA antibody.
  • the parental antibody is an IgG1 antibody.
  • the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • the present invention provides modified antibodies or fragments thereof comprising a peptide tag that is a substrate of a 4′-phosphopantetheinyl transferase, and wherein said peptide tag is inserted between positions 2 and 3 of the V H domain, positions 63 and 64 of the V H domain, positions 64 and 65 of the V H domain, positions 138 and 139 of the CH1 domain, positions 197 and 198 of the CH1 domain, positions 359 and 360 of the CH3 domain, positions 388 and 389 of the CH3 domain, the C-terminus of the CH3 domain (after Lys447), and/or positions 2 and 3 of the V L domain of a parental antibody or fragment thereof.
  • the peptide tag is inserted between amino acid residues 2 and 3 of the VH or VL domain, or between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 164 and 165, or between 165 and 166, or between 194 and 195 of the CH1 domain, or between 388 and 389, or between 445 and 446, or between 446 and 447 of the CH3 domain of a parental antibody or fragment thereof.
  • the peptide tag is inserted between amino acid residue 110 and 111 of the light chain, or between 119 and 120, or between 120 and 121, or between 135 and 136, or between 136 and 137, or between 138 and 139, or between 165 and 166 of the CH1 domain, or between 388 and 389 of the CH3 domain of a parental antibody or fragment thereof.
  • the present invention provides a modified antibody or fragment thereof comprising SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, and/or SEQ ID NO:141.
  • the present invention provides a modified antibody or fragment thereof comprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:139, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:277, SEQ ID NO:348, SEQ ID NO:26
  • the present invention provides a modified antibody or fragment thereof comprising SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:169, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:268, SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:374, or SEQ ID NO:384.
  • said peptide tag is one or more peptides selected from those described in Table 2.
  • the antibody heavy chain and/or light chain (or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its structural loops.
  • the antibodies or antibody fragments contain 2, 4, 6, 8, or more protein tags in its structural loops.
  • said 4′-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a mutant form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • said 4′-phosphopantetheinyl transferase originates from Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima, Clostridium thermocellum , as well as any other mammalian, bacterial or fungal genome.
  • said 4′-phosphopantetheinyl transferase is Sfp and the peptide tag is selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19).
  • the parental antibody is an IgG, IgM, IgE, or IgA antibody.
  • the parental antibody is an IgG1 antibody.
  • the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • the present invention provides modified antibodies or fragments thereof comprising a peptide tag that is a substrate of a 4′-phosphopantetheinyl transferase, and wherein said peptide tag is grafted into a structural loop, or C- or N-terminus of the antibody or fragment thereof.
  • said peptide tag is grafted at amino acid positions from 62 to 64 of the V H domain (mutations at amino acids 62 and 63, and insertion of the rest of the peptide tag between amino acids 63 and 64), at amino acid positions from 62 to 65 of the V H domain (mutations at amino acids 62-64, and insertion of the rest of the peptide tag between amino acids 64 and 65); at amino acid positions from 133 to 139 of the CH1 domain (mutations of amino acids 133-138, and insertion of the rest of the peptide tag between amino acids 138-139), amino acid positions from 189 to 195 of the CH1 domain, and/or amino acid positions from 190 to 198 of the CH1 domain (mutations from amino acids 190-197, and insertion of the rest of the peptide tag between 197 and 198) of a parental antibody or fragment thereof.
  • said peptide tag is one or more peptides selected from those described in Table 2.
  • the modified antibody heavy chain and/or light chain (or fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its structural loops.
  • the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more protein tags in its structural loops.
  • said 4′-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a mutant form thereof that retains the 4′-phosphopantetheinyl transferase activity.
  • said 4′-phosphopantetheinyl transferase originates from Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima, Clostridium thermocellum , as well as any other mammalian, bacterial or fungal genome.
  • said 4′-phosphopantetheinyl transferase is Sfp and the peptide tag is selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19).
  • the parental antibody is an IgG, IgM, IgE, or IgA antibody.
  • the parental antibody is an IgG1 antibody.
  • the parental antibody is an IgG2, IgG3, or IgG4 antibody.
  • the modified antibodies provided herein are engineered to contain one or more orthogonal conjugation sites.
  • orthogonal conjugation sites include, but are not limited to, a substrate of Sfp 4′-phosphopantetheinyl transferase, a substrate of AcpS 4′-phosphopantetheinyl transferase, T. maritima 4′-phosphopantetheinyl transferase, human 4′-phosphopantetheinyl transferase, a lysine, a cysteine, a tyrosine, a histidine, an unnatural amino acid, pyrrolysine and pyrroline-carboxy-lysine.
  • the orthogonal conjugation sites may also be peptide sequences that can be enzymatically or chemically modified, e.g., a tetracysteine tag, a LPXTG-sortase peptide (SEQ ID NO:1057) (X is any amino acid), a biotin acceptor peptide, a CXPXR-aldehyde tag (SEQ ID NO:1058) (X is any amino acid), or a His tag.
  • a tetracysteine tag e.g., a tetracysteine tag, a LPXTG-sortase peptide (SEQ ID NO:1057) (X is any amino acid), a biotin acceptor peptide, a CXPXR-aldehyde tag (SEQ ID NO:1058) (X is any amino acid), or a His tag.
  • engineered antibodies are labeled using the methods of the invention in combination with other conjugation methods known in the art including, but not limited to, chemoselective conjugation through cysteine, lysine, histidine, tyrosine, formyl-glycine, pyrrolysine, pyrroline-carboxylysine and unnatural amino acids.
  • the enzymes Sfp and AcpS are used for orthogonal site-specific labeling of the same or two different labels onto an antibody engineered to contain an S-series peptide (for example, S1, S2, S3, S4, S5, S6 and S7) and an A-series peptide (for example, A1, A-1, A-2, A-3, A-4 and A-6) located in the VH, VL, CH1, CH2, CH3, or C L region of the antibody (see also Table 2).
  • S-series peptide for example, S1, S2, S3, S4, S5, S6 and S7
  • an A-series peptide for example, A1, A-1, A-2, A-3, A-4 and A-6 located in the VH, VL, CH1, CH2, CH3, or C L region of the antibody (see also Table 2).
  • the enzymes Sfp and AcpS are used for orthogonal site-specific labeling of two different labels onto an antibody engineered to contain an ybbR-series peptide (for example, ybbR11, ybbR12 and ybbR13) and an A-series peptide (for example, A1, A-1, A-2, A-3, A-4 and A-6) located in the CH1, CH2, CH3, or C L region of the antibody.
  • an ybbR-series peptide for example, ybbR11, ybbR12 and ybbR13
  • A-series peptide for example, A1, A-1, A-2, A-3, A-4 and A-6 located in the CH1, CH2, CH3, or C L region of the antibody.
  • the enzymes Sfp or AcpS are used for orthogonal site-specific labeling onto an antibody engineered to contain an ybbR-series peptide (for example, ybbR11, ybbR12 and ybbR13) and an A-series peptide (for example, A1, A-1, A-2, A-3, A-4 and A-6) located in the VH, VL, CH1, CH2, CH3, or C L region of the antibody in combination with other conjugation methods.
  • an ybbR-series peptide for example, ybbR11, ybbR12 and ybbR13
  • A-series peptide for example, A1, A-1, A-2, A-3, A-4 and A-6 located in the VH, VL, CH1, CH2, CH3, or C L region of the antibody in combination with other conjugation methods.
  • Such methods include but are not limited to conjugation to lysine, cysteine, tyrosine, histidine, formyl glycine, unnatural amino acids, pyrrolysine and/or pyrroline-carboxy-lysine.
  • Such methods can be used to attached the same or different labels than used for the enzymatic conjugation through Sfp or AcpS.
  • PPTases 4′-phosphopantetheinyl transferase
  • protein having 4′-phosphopantetheinyl transferase activity are used interchangeably and refer to any protein or a fragment thereof, which is capable of transferring a ppan group from a donor molecule, such as coenzyme A (CoA) or an analogue thereof, to a substrate, such as a peptide tag or an acyl carrier protein.
  • a donor molecule such as coenzyme A (CoA) or an analogue thereof
  • PPTases are enzymes which catalyze post-translational modification of carrier proteins associated with fatty acid synthases (FASs), polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These carrier proteins are commonly referred to as ACP, acyl carrier proteins (FASs and PKSs) or to as PCP, peptidyl carrier proteins (NRPSs).
  • ACPs and PCPs consist of about 80 amino acids and are usually integrated as domains in FAS, PKS, or NRPS multienzyme complexes. In some instances, ACPs and PCPs are also found as free-standing autonomously folded proteins.
  • the ACP is essential for fatty acid and polyketide biosynthesis, because it carries the corresponding metabolic intermediates via covalent attachment to its flexible ppan prosthetic group.
  • the PCP carries out the analogous function in nonribosomal peptide synthesis by transporting peptide intermediates between active sites in NRPS multienzyme complexes.
  • PPTases have been classified into three groups, based on sequence and structural similarity and substrate specificity. Members of the first group of PPTases, for example, AcpS of Escherichia coli , are about 120 amino acid residues long, function as homotrimers, and have fairly narrow substrate specificities limited to, for example, to the acyl carrier proteins (ACPs) of type II FAS and PKS systems.
  • ACPs acyl carrier proteins
  • the third group includes PPTases that are attached covalently to the type I FASs, such as those associated with the yeast cytosolic FAS. (see, e.g., Fichtlscherer et al., Eur. J. Biochem., 267:2666-71, 2000).
  • PPTases include naturally occurring proteins having 4′-phosphopantetheinyl transferase activity, including but not limited to, AcpS from E. coli (type I PPTase) and Sfp from B. subtilis (type II PPTase), integrated PPTase domains (type III PPTase) associated with fatty acid synthases (FAS) from S. cerevisiae, S. pombe, C. albacans, E. nidulans , and P. patulum , EntD from E. coli, S. flexneri, S. typhimurium and S. austin , Psf-1 from B. pumilus , Gsp from B.
  • AcpS from E. coli type I PPTase
  • Sfp from B. subtilis
  • type III PPTase integrated PPTase domains associated with fatty acid synthases
  • FOS fatty acid synthases
  • PPTases of the present invention also include proteins having 4′-phosphopantetheinyl transferase activity from species other than the ones described above, as well as those artificially or recombinantly produced proteins, which are capable of 4′-phosphopantetheinylating a peptide moiety described herein.
  • Sfp and AcpS represent two classes of 4′-phosphopantetheinyl transferases that show differences both in their substrate specificity for the carrier protein domains and in their structures (Flugal et al., J. Biol. Chem., 275:959-968, 2000; Lambalot et al., Chem. Biol., 3:923-936, 1996).
  • the Sfp class of pseudodimeric PPTases are about 230 residues in size and the crystal structure of Sfp suggests it has a twofold symmetry with the N— and the C-terminal halves of the molecule adopting similar folds, with the active site of the enzyme at the interface (Hodneland et al., Proc. Natl. Acad.
  • AcpS is about 120 residues in length, about half the size of Sfp, and the crystal structures of AcpS show that the enzyme assembles into trimers and the ACP and CoA binding sites are formed at the interface between each monomer (Reuter et al., Embo. J., 18:6823-6831, 1999; Chirgadze et al., Embo. J., 19:5281-5287, 2000).
  • Sfp exhibits a much broader substrate specificity than AcpS in that Sfp can modify both PCP and ACP domains from nonribosomal peptides synthetases, polyketide synthases, and fatty acid synthases, while AcpS modifies only ACP (Flugel et al., J. Biol. Chem., 275:959-968, 2000; Parris et al., Structure, 8:883-895, 2000; Mofid et al., J. Biol. Chem., 277:17023-17031, 2002).
  • ACP and PCP substrates of both kinds of PPTases adopt similar folds as four-helix bundle proteins with the serine residue to be modified by the ppan prosthetic group at the top of the second alpha-helix, which has been shown to play an important role for interacting with Sfp and AcpS (Hodneland et al., Proc. Natl. Acad. Sci. USA, 99:5048-5052, 2002; Chirgadze et al., Embo. J., 19:5281-5287, 2000; Quadri et al., Biochem., 37:1585-1595, 1998; L 1 et al., Biochem., 42:4648-4657, 2003).
  • ybbR13 is an 11 amino acid residue peptide, which is a substrate of Sfp (J. Yin et al., Proc. Natl. Acad. Sci. USA, 102:15815-15820, 2005; Z. Zhou et al., ACS Chem. Biol., 2:337-346, 2007; Z. Zhou et al., J. Am. Chem. Soc., 130: 9925-9930, 2008).
  • the ybbR13 peptide (DSLEFIASKLA (SEQ ID NO:9) was isolated from a phage displayed library of the B. subtilis genome (J.
  • ybbR13 peptide A part of the sequence of the ybbR13 peptide is derived from a B. subtilis open reading frame, called ybbR, and it includes the (H/D)S(L/I) tri-peptide sequence at the N-terminus, which is conserved in known substrates of PPTases such as ACPs, PCPs, and aryl carrier proteins (ArCPs).
  • the ybbR peptide does not include the amino acid sequence, DxFFxxLGG (SEQ ID NO:1059) at its N-terminus, which is found to be conserved in PCPs.
  • Exemplary S series of peptides include, but are not limited to, S6, which is an efficient substrate for Sfp, and exemplary A series of peptides include, but are not limited to, A1, which is an efficient substrate for AcpS. Both S6 and A1 peptides are 12 amino acid residues in length.
  • peptide substrates examples are listed in Table 2 below.
  • these short peptide tags can be used for the site-specific labeling of target proteins (including antibodies) in reactions catalyzed by PPTases.
  • a pairing of peptide tags and respective PPTases described herein, e.g., ybbR13/Sfp or S6/Sfp and A1/AcpS can also be used for orthogonal site-specific labeling of one (or multiple) target proteins, e.g., in cell lysates or on the surface of live cells.
  • the present invention provides engineered antibodies which contain one or more of the peptide tags listed in Table 2, and methods of labeling such antibodies, e.g., conjugating with a cytotoxin.
  • the labeling chemistry is illustrated below and in the Examples.
  • the modified antibody or fragment thereof provided herein are site-specifically labeled by post-translational modification of the short peptide tag (inserted or grafted or combination thereof) using PPTases or mutants thereof, including, but not limited to, Sfp, AcpS, human PPTase or T. maritima PPTase.
  • PPTases or mutants thereof including, but not limited to, Sfp, AcpS, human PPTase or T. maritima PPTase.
  • Such post-translational modifications involve a PPTase catalyzed reaction between a conserved serine residue in the short peptide tag and a 4′-phosphopantetheinyl (ppan) group of coenzyme A (CoA) or a coenzyme A analogue.
  • the ppan prosthetic group of CoA, or modified ppan prosthetic group of the CoA analogue is attached to the short peptide tag through the formation of a phosphodiester bond with the hydroxyl group of the conserved serine residue of the short peptide tag which has been incorporated (i.e. inserted or grafted or combination thereof) into the antibody.
  • the ppan or modified ppan is linked to a terminal group (TG) and the formation of the phosphodiester bond thereby conjugates the terminal group (TG) to the modified antibody or fragment thereof via a linker which includes the ppan or modified ppan moiety.
  • the modified antibodies or fragment thereof provided herein are labeled by a one-step method wherein the post-translational modification occurs by reacting a CoA linked to a terminal group (TG), or a CoA analogue linked to a terminal group (TG), with the conserved serine of the short peptide tag engineered into the antibody, as shown in Schemes (Ia)-(Ic) below.
  • the modified antibodies or fragment thereof are labeled by a two-step method wherein the post-translational modification involves first reacting an activated CoA or an activated CoA analogue with the conserved serine of the short peptide tag engineered into the antibody, followed by reacting a functionalized terminal group (TG) with the reactive group on the activated CoA or an activated CoA.
  • TG functionalized terminal group
  • the modified antibodies or fragment thereof are labeled by a three-step method, wherein the post-translational modification involves first reacting a CoA having a protected ppan prosthetic group, or a CoA analogue having protected ppan prosthetic group, with the conserved serine of the short peptide tag engineered into the antibody, thereby attaching the CoA or CoA analogue to the antibody.
  • the protected ppan prosthetic group is deprotected thereby generating a reactive functional group on the protected ppan prosthetic group.
  • this reactive functional group is linked to a terminal group (TG), thereby attaching the terminal group to the modified antibody or fragment thereof.
  • TG terminal group
  • R 2 is H or P( ⁇ O)(OH) 2 ;
  • L 1 is —C( ⁇ O)—NH—CH 2 —CH 2 —S-[L 2 -L 3 -L 4 -TG]. (Portions of these formulas depicted in brackets such as [L 2 -L 3 -L 4 -TG] are added to the formula being described in order to identify which open valence of the formula is attached to the bracket-enclosed part of the remainder of the structure.)
  • L 2 is selected from:
  • L 3 is selected from —(CH 2 ) 2-6 —C( ⁇ O)-[L 4 -TG]; —(CH 2 ) 2-6 —NH-[L 4 -TG]; (CH 2 ) 2-6 —S-[L 4 -TG]; —(CH 2 ) 2-6 —Z-[L 4 -TG]; and —(CH 2 ) 2-6 —Z—C( ⁇ O)-[L 4 -TG], where Z is O, NH or S.
  • L 4 is a bond or a val-cit linker of this formula:
  • L 3 is preferably —(CH 2 ) 2-6 —C( ⁇ O),
  • TG is a maytansinoid such as DM1 or DM4, or a dolostatin 10 compound, e.g. auristatins MMAF or MMAE, or a calicheamicin such as N-acetyl- ⁇ -calicheamicin, or a label or dye such as rhodamine or tetramethylrhodamine.
  • a dolostatin 10 compound e.g. auristatins MMAF or MMAE
  • a calicheamicin such as N-acetyl- ⁇ -calicheamicin
  • a label or dye such as rhodamine or tetramethylrhodamine.
  • linker is any chemical moiety that is capable of linking an antibody or a fragment thereof to a terminal group.
  • Linkers can be susceptible to cleavage, such as, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.
  • linkers can be substantially resistant to cleavage.
  • a linker may or may not include a self-immolative spacer.
  • Non-limiting examples of the non-enzymatically cleavable linkers as used herein to conjugate a terminal group (TG) to the modified antibodies or fragment thereof provided herein include, acid-labile linkers, linkers containing a disulfide moiety, linkers containing a triazole moiety, linkers containing a hydrazine moiety, linkers containing a thioether moiety, linkers containing a diazo moiety, linkers containing an oxime moiety, linkers containing an amide moiety and linkers containing an acetamide moiety.
  • Non-limiting examples of the enzymatically cleavable linkers as used herein to conjugate a terminal group (TG) to the modified antibodies or fragment thereof provided herein include, but are not limited to, linkers which are cleaved by a protease, linkers which are cleaved by an amidase, and linkers which are cleaved by ⁇ -glucuronidase.
  • such enzyme cleavable linkers are linkers which are cleaved by cathepsin, including cathepsin Z, cathepsin B, cathepsin H and cathepsin C.
  • the enzymatically cleavable linker is a dipeptide cleaved by cathepsin, including dipeptides cleaved by cathepsin Z, cathepsin B, cathepsin H or cathepsin C.
  • the enzymatically cleavable linker is a cathepsin B-cleavable peptide linker.
  • the enzymatically cleavable linker is a cathepsin B-cleavable dipeptide linker. In certain embodiments the enzymatically cleavable linker is a cathepsin B-cleavable dipeptide linker is valine-citrulline or phenylalanine-lysine.
  • Other non-limiting examples of the enzymatically cleavable linkers as used herein conjugate a terminal group (TG) to the modified antibodies or fragment thereof provided herein include, but are not limited to, linkers which are cleaved by ⁇ -glucuronidase, e.g.,
  • Self-immolative spacers are bifunctional chemical moieties covalently linked at one termini to a first chemical moiety and at the other termini to a second chemical moiety, thereby forming a stable tripartate molecule.
  • self-immolative spacers Upon cleavage of a bond between the self-immolative spacer and the first chemical moiety, self-immolative spacers undergoing rapid and spontaneous intramolecular reactions and thereby separate from the second chemical moiety.
  • These intramolecular reactions generally involve electronic rearrangements such as 1,4, or 1,6, or 1,8 elimination reactions or cyclizations to form highly favored five- or six-membered rings.
  • the first moiety is an enzyme cleavable linker and this cleavage results from an enzymatic reaction, while in other embodiments the first moiety is an acid labile linker and this cleavage occurs due to a change in pH.
  • the second moiety is the “Label” group as defined herein.
  • cleavage of the first moiety from the self-immolative spacer results from cleavage by a proteolytic enzyme, while in other embodiments it results from cleaved by a hydrolase.
  • cleavage of the first moiety from the self-immolative spacer results from cleavage by a cathepsin enzyme.
  • the enzyme cleavable linker is a peptide linker and the self-immolative spacer is covalently linked at one of its ends to the peptide linker and covalently linked at its other end to a drug moiety.
  • This tripartite molecule is stable and pharmacologically inactive in the absence of an enzyme, but which is enzymatically cleavable by enzyme at the bond covalently linking the spacer moiety and the peptide moiety.
  • the peptide moiety is cleaved from the tripartate molecule which initiates the self-immolating character of the spacer moiety, resulting in spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.
  • Non-limiting examples of the self-immolative spacer optionally used in the conjugation of a terminal group (TG) to the modified antibodies or fragment thereof provided herein include, but are not limited to, moieties which include a benzyl carbonyl moiety, a benzyl ether moiety, a 4-aminobutyrate moiety, a hemithioaminal moiety or a N-acylhemithioaminal moiety.
  • self-immolative spacers include, but are not limited to, p-aminobenzyloxycarbonyl groups, aromatic compounds that are electronically similar to the p-aminobenzyloxycarbonyl group, such as 2-aminoimidazol-5-methanol derivatives and ortho or para-aminobenzylacetals.
  • self-immolative spacers used herein which undergo cyclization upon amide bond hydrolysis include substituted and unsubstituted 4-aminobutyric acid amides and 2-aminophenylpropionic acid amides.
  • the self-immolative spacer is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-immolative spacer
  • n 1 or 2.
  • the self-immolative spacer is
  • n 1 or 2.
  • the self-immolative spacer is
  • n 1 or 2.
  • the self-immolative spacer is
  • n 1 or 2.
  • the self-immolative spacer is
  • n 1 or 2.
  • Scheme (Ib) illustrates the post-translational modification of the modified antibodies or fragment thereof provided herein wherein the Linker Unit (LU) is -L 1 -L 2 -L 3 -L 4 -.
  • R 2 , L 1 , L 2 , L 3 , L 4 and TG are as defined herein.
  • the CoA analogues of Scheme (Ia) and Scheme (Ib) may be obtained by total chemical synthesis, however the CoA analogues of Scheme (Ia) and Scheme (Ib) are preferably obtained by a chemoenzymatic process wherein pantetheine analogues are chemically synthesized and then biosynthetically converted into the corresponding CoA analogue (see Kristine M. Clarke et al., “In Vivo Reporter Labeling of Proteins via Metabolic Delivery of Coenzyme A Analogues”, J. Am. Chem. Soc., 2005, 127, p. 11234-11235 and Jordan L.
  • the biosynthetic conversion occurs “in-vivo”, wherein the pantetheine analogue enters a cell from the surrounding media whereby once inside the cell it is converted by the CoA enzymatic pathway into the corresponding CoA analogue.
  • E. coli is used for the biosynthetic conversion of pantetheine analogues into the corresponding CoA analogues, wherein the pantetheine analogue enters E.
  • the pantetheine analogue is initially phosphorylated by the pantothenate kinase (PanK or CoaA) using adenosine-5′-triphosphate (ATP), then adenylated by the phosphopantetheine adenylyltransferase (PPAT or CoaD) to give the dephospho-CoA analogue and then further phosphorylated by the dephosphocoenzyme A kinase (DPCK or CoaE) to yield the CoA analogue.
  • PanK or CoaA pantothenate kinase
  • ATP adenosine-5′-triphosphate
  • PPAT or CoaD phosphopantetheine adenylyltransferase
  • DPCK or CoaE dephosphocoenzyme A kinase
  • the biosynthetic conversion occurs “in-vitro”, wherein the enzymatic CoA pathway is reconstituted with the pantetheine analogue, whereby it is converted “in-vitro” by the reconstituted CoA enzymatic pathway into the corresponding CoA analogue.
  • the reconstituted CoA enzymatic pathway is the E. coli CoA enzymatic pathway, wherein the pantetheine analogue is initially phosphorylated by CoaA and ATP, then adenylated by CoaD to give the dephospho-CoA analogue and then further phosphorylated by CoaE to yield the CoA analogue.
  • the Linker Unit (LU) is —C( ⁇ O)NH(CH 2 ) 2 S-L 2 -L 3 -L 4 - and R 2 is —P( ⁇ O)(OH) 2 , and in such an embodiment the terminal group is linked to CoA.
  • Scheme (Ic) illustrates the post-translational modification of the modified antibodies or fragment thereof provided herein for the specific embodiment wherein the PPTase catalyzes the reaction between the conserved serine residue in the incorporated short peptide tag and a terminal group (TG) linked to coenzyme A (CoA):
  • the modified antibodies or fragment thereof provided herein are site-specifically labeled by a one-step method as shown in Scheme (Ia), Scheme (Ib) and Scheme (Ic), wherein a terminal group linked to CoA or a CoA analogue reacts with the conserved serine of the short peptide tag engineered into the antibody.
  • the one step method includes the steps of:
  • the terminal group (TG) is thereby conjugated to the modified antibody or fragment thereof via a linker having the structure according to Formula (I-a).
  • the linker of Formula (I-a) is attached to the small peptide tag by a phosphodiester bond formed between the 4′-phosphopantetheinyl moiety and the hydroxyl group of the conserved serine residue of the short peptide tag engineered into the antibody:
  • the one step method includes the steps of:
  • R 2 , L 1 , L 2 , L 3 , L 4 and TG are as defined herein.
  • the terminal group is thereby attached to the modified antibody or fragment thereof via a linker having the structure according to Formula (I-b).
  • the linker of Formula (I-b) is attached to the small peptide tag by a phosphodiester bond formed between the 4′-phosphopantetheinyl moiety and the hydroxyl group of the conserved serine residue of the short peptide tag engineered into the antibody:
  • the one step method includes the steps of:
  • L 2 , L 3 , L 4 and TG are as defined herein.
  • the terminal group is thereby attached to the modified antibody or fragment thereof via a linker having the structure according to Formula (I-c).
  • the linker of Formula (I-c) is attached to the small peptide tag by a phosphodiester bond formed between the 4′-phosphopantetheinyl moiety and the hydroxyl group of the conserved serine residue of the short peptide tag engineered into the antibody:
  • L 2 , L 3 and L 4 are as defined herein and the * denotes that the 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • the modified antibody or fragment thereof is contacted with a compound having the structure of Formula (A), Formula (B) or Formula (C) and a 4′-phosphopantetheinyl transferase enzyme that is co-expressed in the same cell as the expressed modified antibody or fragment thereof.
  • the modified antibody or fragment thereof is contacted in the cell culture media with a compound having the structure of Formula (A), Formula (B) or Formula (C) and 4′-phosphopantetheinyl transferase enzyme produced in the same or in another cell.
  • the 4′-phosphopantetheinyl transferase enzyme is immobilized on solid support.
  • the solid support is optionally comprised of a polymer on a bead or a column.
  • L 1 is -A 1 X 2 —
  • L 2 is a bond
  • L 3 is a bond
  • L 4 is -A 4 -
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S—
  • L 1 is -A 1 X 2 —, L 2 is a bond, L 3 is a bond, L 4 is -A 4 -, A 1 is —C( ⁇ O)NH(CH 2 ) n S—, A 4 is —(CH 2 ) n NHC( ⁇ O)—;
  • X 2 is
  • TG is a fluorescent probe.
  • L 1 is -A 1 X 2 —
  • L 2 is a bond
  • L 3 is a bond
  • L 4 is -A 4 -
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S—
  • a 4 is —(CH 2 ) n C( ⁇ O)—
  • X 2 is
  • L 1 is -A 1 X 2 —, L 2 is a bond, L 3 is a bond, L 4 is -A 4 -, A 1 is —C( ⁇ O)NH(CH 2 ) n S—, A 4 is —(CH 2 ) n C( ⁇ O)—;
  • X 2 is
  • TG is a drug moiety.
  • L 1 is -A 1 X 2 —
  • L 2 is -A 2 -
  • L 3 is -A 3 -
  • L is
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S—
  • a 2 is —(CH 2 ) n C( ⁇ O)
  • a 3 is
  • L 1 is -A 1 X 2 —
  • L 2 is -A 2 -
  • L 3 is -A 3 -
  • L 4 is
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S—
  • a 2 is —(CH 2 ) n C( ⁇ O)
  • a 3 is
  • TG is a drug moiety.
  • L 1 is a -A 1 X 2 —
  • L 2 is a bond-
  • L 3 is -A 3 -
  • L 4 is a bond
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S—
  • a 3 is —(CH 2 ) n C( ⁇ O)—
  • X 2 is —(CH 2 ) n C( ⁇ O)NH—.
  • L 1 is a -A 1 X 2 —
  • L 2 is a bond-
  • L 3 is -A 3 -
  • L 4 is a bond
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S—
  • a 3 is —(CH 2 ) n C( ⁇ O)—
  • X 2 is —(CH 2 ) n C( ⁇ O)NH—
  • TG is a drug moiety.
  • L 1 is a -A 1 X 2 —
  • L 2 is a bond
  • L 3 is -A 3 -
  • L 4 is a bond
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S
  • a 3 is —(CH 2 ) n C( ⁇ O)—
  • X 2 is —CHR 4 (CH 2 ) n C( ⁇ O)NH—
  • R 4 is —C( ⁇ O)OH.
  • L 1 is a -A 1 X 2 —
  • L 2 is a bond
  • L 3 is -A 3 -
  • L 4 is a bond
  • a 1 is —C( ⁇ O)NH(CH 2 ) n S
  • a 3 is —(CH 2 ) n C( ⁇ O)—
  • X 2 is —CHR 4 (CH 2 ) n C( ⁇ O)NH—
  • R 4 is —C( ⁇ O)OH
  • TG is a drug moiety.
  • L 1 is -A 1 X 2 —, where A 1 is —C( ⁇ O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C( ⁇ O)NH—; L 2 is a bond; L 3 is a bond, and L 4 is -A 4 - wherein A 4 is —(CH 2 ) n NHC( ⁇ O)—.
  • L 1 is -A 1 X 2 —, wherein A 1 is —C( ⁇ O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C( ⁇ O)NH—; L 2 is a bond; L 3 is a bond; L 4 is -A 4 -, wherein A 4 is —(CH 2 ) n C( ⁇ O)—.
  • L 1 is -A 1 X 2 —, wherein A 1 is —C( ⁇ O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C( ⁇ O)NH—;
  • L 2 is -A 2 -, wherein A 2 is —(CH 2 ) n C( ⁇ O;
  • L 3 is -A 3 -, wherein A 3 is
  • L 1 is a -A 1 X 2 —, wherein A 1 is —C( ⁇ O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C( ⁇ O)NH—; L 2 is a bond-; L 3 is -A 3 -, wherein A 3 is —(CH 2 ) n C( ⁇ O)—, and L 4 is a bond.
  • the modified antibodies or fragment thereof provided herein are site-specifically labeled by a two-step method, wherein, in the first step the ppan prosthetic group of CoA, or modified ppan prosthetic group of the CoA analogue, which contain a functional group (R 1 ), is attached to the short peptide tag by a phosphodiester bond formed between the 4′-phosphopantetheinyl moiety and the hydroxyl group of the conserved serine residue of the short peptide tag which has been incorporated into the antibody.
  • a terminal group (TG) linked, or directly attached to, a group which is reactive with the functional group (R 1 ) is reacted with the functional group (R 1 ) on the ppan prosthetic group of CoA, or on the modified ppan prosthetic group of the CoA analogue, thereby directly attaching the terminal group to the modified antibody or fragment thereof or attaching the terminal group to the modified antibody or fragment thereof via a Linker Unit (LU).
  • LU Linker Unit
  • Terminal group is attached to the modified antibody or fragment thereof via a linker having the structure according to Formula (IIb):
  • a 1 , X 2 , L 2 , L 3 and L 4 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • the Two-Step Method of Scheme (IIb) includes the steps of:
  • L 1 , A 2 , X 2 , L 3 and L 4 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • the Two-Step Method of Scheme (IIc) includes the steps of:
  • L 1 , L 2 , A 3 , X 2 and L 4 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • the Two-Step Method of Scheme (IId) includes the steps of:
  • L 1 , L 2 , L 3 , A 4 and X 2 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • the modified antibody or fragment thereof is contacted with a compound having the structure of Formula (D), Formula (E), Formula (F) or Formula (G) and a 4′-phosphopantetheinyl transferase enzyme that is co-expressed in the same cell as the expressed modified antibody or fragment thereof.
  • the modified antibody or fragment thereof is contacted in the cell culture media with a compound having the structure of Formula (D), Formula (E), Formula (F) or Formula (G) and 4′-phosphopantetheinyl transferase enzyme produced in the same or in another cell.
  • the 4′-phosphopantetheinyl transferase enzyme is immobilized on solid support.
  • the solid support is optionally comprised of a polymer on a bead or a column.
  • Table 4 shows certain embodiments of the activated 4′-phosphopantetheinyl groups of Formula (D-a) and compounds of Formula (II-a) used in the Two-step methods and the Three-step methods described herein and the resulting modified serine located in the modified antibody or fragment thereof.
  • a 1 , L 2 , L 3 , L 4 , R 5 , R 6 , R 7 , R 8 and TG are as defined herein, and Y is
  • Table 5 shows certain embodiments of the activated 4′-phosphopantetheinyl groups of Formula (E-a) and compounds of Formula (II-c) used in the Two-step methods and the Three-step methods described herein and the resulting modified serine located in the modified antibody or fragment thereof.
  • L 1 , A 2 , L 3 , L 4 , R 5 , R 6 , R 7 , R 8 and TG are as defined herein, and Y is
  • Table 6 shows certain embodiments of the activated 4′-phosphopantetheinyl groups of Formula (F-a) and compounds of Formula (II-e) used in the Two-step methods and the Three-step methods described herein and the resulting modified serine located in the modified antibody or fragment thereof.
  • L 1 , L 2 , A 3 , L 4 , R 5 , R 6 , R 7 , R 8 and TG are as defined herein, and Y is
  • Table 7 shows certain embodiments of the activated 4′-phosphopantetheinyl groups of Formula (G-a) and compounds of Formula (II-g) used in the Two-step methods and the Three-step methods described herein and the resulting modified serine located in the modified antibody or fragment thereof.
  • L 1 , L 2 , L 3 , A 4 , R 5 , R 6 , R 7 , R 8 and TG are as defined herein, and Y is
  • the modified antibodies or fragment thereof provided herein are site-specifically labeled by a three-step method, wherein, in the first step a protected ppan prosthetic group of CoA, or a protected modified ppan prosthetic group of the CoA analogue, is attached to the short peptide tag by a phosphodiester bond formed between the 4′-phosphopantetheinyl moiety and the hydroxyl group of the conserved serine residue of the short peptide tag incorporated into the antibody.
  • the protected ppan prosthetic group of CoA, or protected modified ppan prosthetic group of the CoA analogue is deprotected; thereby generating a reactive functional group (R 1 ).
  • a terminal group (TG) linked, or directly attached to, a group which is reactive with the functional group (R 1 ) is reacted with the functional group (R 1 ) on the ppan prosthetic group of CoA, or on the modified ppan prosthetic group of the CoA analogue, thereby directly attaching the terminal group to the modified antibody or fragment thereof or attaching the terminal group to the modified antibody or fragment thereof via a Linker Unit (LU).
  • LU Linker Unit
  • PG is a protecting group and X, R 1 , R 2 , A 1 , L 2 , L 3 , L 4 and TG are as defined herein.
  • a 1 , X 2 , L 2 , L 3 and L 4 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • PG is a protecting group and X, R 1 , R 2 , L 1 , A 2 , L 3 , L 4 and TG are as defined herein.
  • L 1 , A 2 , X 2 , L 3 and L 4 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • PG is a protecting group and X, R 1 , R 2 , L 1 , L 2 , L 3 , L 4 and TG are as defined herein.
  • L 1 , L 2 , A 3 , X 2 and L 4 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • R 1 , R 2 , L 1 , L 2 , L 3 , L 4 and TG are as defined herein.
  • L 1 , L 2 , L 3 , A 4 and X 2 are as defined herein and the * denotes that the modified 4′-phosphopantetheinyl moiety is attached to the small peptide tag.
  • Scheme (IIIe) shows a certain embodiment of the Three-Step Method where the modified antibodies or fragment thereof provided herein are site-specifically labeled by a CoA analogue where the thiol of the 4′-phosphopantetheinyl prosthetic group is protected.
  • step 1 the protected CoA analogue reacts with the conserved serine of the short peptide tag engineered into the antibody thereby attaching the prosthetic group containing the protected thiol to the short peptide tag through the formation of a phosphodiester bond with the hydroxyl group of the conserved serine residue of the short peptide tag.
  • the thiol protecting group is removed and the resulting modified antibody or fragment thereof having a pendant 4′-phosphopantetheinyl group is reacted with a thiol reactive group linked to a terminal group (TG).
  • X SH , protecting group (PG), R 2 , A 2 , L 3 , L 4 and TG are as defined herein.
  • Scheme (IIIf) shows a certain embodiment of the Three-Step Method where the modified antibodies or fragment thereof provided herein are site-specifically labeled using a CoA where the thiol of the 4′-phosphopantetheinyl prosthetic group is protected.
  • step 1 the protected CoA reacts with the conserved serine of the short peptide tag engineered into the antibody thereby attaching the prosthetic group containing the protected thiol to the short peptide tag through the formation of a phosphodiester bond with the hydroxyl group of the conserved serine residue of the short peptide tag.
  • the thiol protecting group is removed and the resulting modified antibody or fragment thereof having a pendant 4′-phosphopantetheinyl group is reacted with a thiol reactive group linked to a terminal group (TG).
  • the thiol protecting group includes, but is not limited to, acetyl, acetamidomethyl, benzyl, 4-methylbenzyl, 4-methoxybenzyl, trityl, methoxytrityl, t-butyl, t-butylthiol and 3-nitro-2-pyridinesulphenyl.
  • the thiol reactive group of Scheme (IIIe) and Scheme (IIIf) includes, but is not limited to, maleimide, a haloacetyl, a haloacetamide, a pyridyldisulfide and a vinyl sulfone.
  • the Three-Step Method of Scheme (IIIf) includes the steps of:
  • X SH is a thiol reactive group including, but not limited to, a maleimide, a haloacetyl, a haloacetamide, a pyridyldisulfide and a vinyl sulfone.
  • a 2 , L 3 , L 4 and TG are as defined herein.
  • the terminal group is attached to the modified antibody or fragment thereof via a linker having the structure according to Formula (III-a):
  • X 2 is a group formed by reaction of X SH and the pendant thiol, including, but not limited to,
  • X SH -L 2 -L 3 -L 4 -TG is
  • the modified antibody or fragment thereof is contacted with a compound having the structure of Formula (H), Formula (J), Formula (K) or Formula (L) and a 4′-phosphopantetheinyl transferase enzyme that is co-expressed in the same cell as the expressed modified antibody or fragment thereof.
  • the modified antibody or fragment thereof is contacted in the cell culture media with a compound having the structure of Formula (H), Formula (J), Formula (K) or Formula (L) and 4′-phosphopantetheinyl transferase enzyme co-expressed by the same or another cell.
  • the 4′-phosphopantetheinyl transferase enzyme is immobilized on solid support.
  • the solid support is optionally comprised of a polymer on a bead or a column.
  • the modified antibody or fragment thereof will be contacted with a 4′-phosphopantetheinyl transferase enzyme that is coexpressed in the same cell.
  • the thiol protected coenzyme A is acetyl-coenzyme A.
  • the modified antibody or fragment thereof is contacted in the cell culture media with the thiol protected coenzyme A and a 4′-phosphopantetheinyl transferase enzyme co-expressed by the same or another cell.
  • the 4′-phosphopantetheinyl transferase enzyme is immobilized on solid support.
  • the solid support is optionally comprised of a polymer on a bead or a column.
  • the modified antibody or fragment thereof is contacted, depending on the Method used, with a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or Formula (L) and a 4′-phosphopantetheinyl transferase enzyme at temperatures between 0 and 37 degree Celsius in buffer or media adjusted to pH values between 3 and 10, preferably between 7 and 9 and most preferably around 8, for reaction times between 5 mins and 48 hours.
  • the modified antibody or fragment thereof is contacted, depending on the Method used, with a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or Formula (L) in the presence of 4′-phosphopantetheinyl transferase in solution.
  • the modified antibody or fragment thereof is contacted, depending on the Method used, with a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or Formula (L) in the presence of 4′-phosphopantetheinyl transferase in cell media.
  • the modified antibody or fragment thereof is contacted, depending on the Method used, with a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or Formula (L) in the presence of 4′-phosphopantetheinyl transferase inside a cell.
  • the modified antibody or fragment thereof is contacted, depending on the Method used, with a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or Formula (L) in the presence of 4′-phosphopantetheinyl transferase, wherein the 4′-phosphopantetheinyl transferase is immobilized on a surface.
  • the surface is polymer bead.
  • the modified antibody or fragment thereof is contacted, depending on the Method used, with a compound having the structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or Formula (L) in the presence of 4′-phosphopantetheinyl transferase, wherein the modified antibody or fragment thereof is immobilized on a surface.
  • the surface is polymer bead.
  • the modified antibody or fragment thereof provided herein are labeled with a terminal group (“TG”)-to-antibody ratio of 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modified antibody or fragment thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 short peptide tags located in the structural loop of the antibody and where the short peptide tags are substrates of Sfp 4′-phosphopantetheinyl transferase, AcpS 4′-phosphopantetheinyl transferase, T. maritima 4′-phosphopantetheinyl transferase, C. thermocellum 4′-phosphopantetheinyl transferase, human 4′-phosphopantetheinyl transferase, or a mutant form thereof.
  • TG terminal group
  • a TG-to-antibody ratio of 4 is achieved by conjugating the terminal group to four copies of inserted S6 tags, or to four copies of inserted ybbR tags or to four copies of inserted A1 tags, or to a combination of two copies of inserted S6 tags and two copies of inserted ybbR tags.
  • the modified antibodiesor fragment thereof provided herein are labeled with two different terminal groups using two different peptide tags and two different 4′-phosphopantetheinyl transferases.
  • two copies of the A1 tag are conjugated to a first terminal group using the AcpS 4′-phosphopantetheinyl transferase.
  • a second terminal group is attached to two copies of an S6 tag using the Sfp 4′-phosphopantetheinyl transferase (see, e.g., Zhou et al., ACS Chem. Biol. 2:337-346, 2007).
  • the modified antibodies or fragment thereof provided herein are labeled with a terminal group (TG)-to-antibody ratio (e.g., DAR) of 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modified antibody or fragment thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 short peptide tags located in the structural loop of the antibody and where the short peptide tags are substrates of Sfp 4′-phosphopantetheinyl transferase, AcpS 4′-phosphopantetheinyl transferase, T. maritima 4′-phosphopantetheinyl transferase, C.
  • TG terminal group
  • DAR terminal group-to-antibody ratio
  • thermocellum 4′-phosphopantetheinyl transferase human 4′-phosphopantetheinyl transferase, or a mutant form thereof.
  • a TG-to-antibody ratio of 4 is achieved by conjugating a drug moiety to four copies of inserted S6 tags, or to four copies of inserted ybbR tags, or to four copies of inserted A1 tags, or to a combination of two copies of inserted S6 tags and two copies of inserted ybbR tags.
  • the modified antibodies or fragment thereof provided herein are labeled with two different drug moieties using two different peptide tags and two different 4′-phosphopantetheinyl transferases.
  • two copies of the A1 tag are conjugated to a first drug moiety using the AcpS 4′-phosphopantetheinyl transferase. Then a second drug moiety is attached to two copies of an S6 tag using the Sfp 4′-phosphopantetheinyl transferase (see, e.g., Zhou et al., ACS Chem. Biol. 2:337-346, 2007).
  • the present invention provides site-specific labeled immunoconjugates.
  • the immunoconjugates of the invention may comprise modified antibodies or fragments thereof that further comprise modifications to framework residues within V H and/or V L , e.g. to improve the properties of the antibody.
  • framework modifications are made to decrease the immunogenicity of the antibody.
  • one approach is to “back-mutate” one or more framework residues to the corresponding germline sequence.
  • an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • somatic mutations can be “back-mutated” to the germline sequence by, for example, site-directed mutagenesis.
  • Such “back-mutated” antibodies are also intended to be encompassed by the invention.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.
  • antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • modifications within the Fc region typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH 2 —CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in, e.g., U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer et al.
  • one or more amino acids of an antibody or fragment thereof of the present invention are replaced by one or more allotypic amino acid residues, such as those shown in FIG. 4 for the IgG1 subclass and the kappa isotype.
  • Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgG1, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc ⁇ receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta.
  • the binding sites on human IgG1 for Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).
  • the glycosylation of an antibody is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for “antigen.”
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • Such an approach is described in, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation.
  • glycoprotein-modifying glycosyl transferases e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • the antibody is modified to increase its biological half-life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward.
  • the antibody can be altered within the CH1 or C L region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.
  • the present invention provides site-specific labeling methods, modified antibodies and fragments thereof, and immunoconjugates prepared accordingly.
  • a modified antibody or fragments thereof can be conjugated to a label, such as a drug moiety, e.g., an anti-cancer agent, an autoimmune treatment agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent.
  • a label such as a drug moiety, e.g., an anti-cancer agent, an autoimmune treatment agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent.
  • An antibody or fragments thereof can also be conjugated using several identical or different labeling moieties combining the methods of the invention with other conjugation methods.
  • the terminal group of the immunoconjugates of the present invention is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an Eg5 inhibitor, an HDAC inhibitor, a RNA polymerase inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.
  • a V-ATPase inhibitor a HSP90 inhibitor
  • an IAP inhibitor an mTor inhibitor
  • modified antibodies or antibody fragments of the present invention may be conjugated to a therapeutic moiety or drug moiety that modifies a given biological response.
  • Therapeutic moieties or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response modifier such as, for example, a lymphokine.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response
  • the modified antibodies or antibody fragments of the present invention are conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • a therapeutic moiety such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • cytotoxin include but not limited to, taxanes (see, e.g., International (PCT) Patent Application Nos.
  • DNA-alkylating agents e.g., CC-1065 analogs
  • anthracyclines e.g., tubulysin analogs
  • duocarmycin analogs e.g., auristatin E
  • auristatin F e.g., maytansinoids
  • cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et al., Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et al., J. Antibiot.
  • colchicin colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, anti-metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thioepa chloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • An example of a calicheamicin antibody conjugate is commercially available (MylotargTM; Wyeth-Ayerst).
  • modified antibodies or fragments thereof can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, referred to as radioimmunoconjugates.
  • radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine 131 , indium 111 , yttrium 90 , and lutetium 177 . Methods for preparing radioimmunoconjugates are established in the art.
  • radioimmunoconjugates are commercially available, including ZevalinTM (DEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention.
  • the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • linker molecules are commonly known in the art and described in Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.
  • the present invention further provides modified antibodies or fragments thereof that specifically bind to an antigen conjugated to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the invention provides fusion proteins comprising an antibody fragment described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab) 2 fragment, a V H domain, a V H CDR, a V L domain or a V L CDR) and a heterologous protein, polypeptide, or peptide.
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol.
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • a polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the modified antibodies or fragments thereof of the present invention can be conjugated to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide (SEQ ID NO:1106), such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., (1984) Cell 37:767), and the “FLAG” tag (A. Einhauer et al., J. Biochem. Biophys. Methods 49: 455-465, 2001).
  • HA hemagglutinin
  • FLAG A. Einhauer et al., J. Biochem. Biophys. Methods 49: 455-465, 2001.
  • antibodies or antibody fragments can also be conjugated to tumor-penetrating peptides in order to enhance their efficacy.
  • modified antibodies or antibody fragments of the present invention are conjugated to a diagnostic or detectable agent.
  • immunoconjugates can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, umbelliferone, fluorescein, fluorescein isothiocyan
  • Modified antibodies or antibody fragments of the invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • compositions including immunoconjugates are mixed with a pharmaceutically acceptable carrier or excipient.
  • the compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynacomastica, and endometriosis).
  • cancer breast cancer, colorectal cancer, lung cancer, multiple myeloma
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al.
  • an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects.
  • the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.
  • compositions comprising antibodies or fragments thereof of the invention can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week.
  • Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation.
  • a specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
  • the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.
  • the dosage of the antibodies or fragments thereof of the invention may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
  • Doses of the immunoconjugates the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In a specific embodiment, does of the immunoconjugates of the invention are repeated every 3 weeks.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).
  • the route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc.
  • composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety.
  • a composition of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • a composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the immunoconjugates of the invention is administered by infusion.
  • the immunoconjugates of the invention is administered subcutaneously.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989).
  • Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7 1:105, 1989; U.S. Pat. No.
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).
  • the immunoconjugates of the invention are administered topically, they can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995).
  • viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed.
  • Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts
  • Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • humectants can also be added to
  • compositions comprising the immunoconjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops.
  • prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a second therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation
  • a second therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation
  • An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.
  • Additional therapies which can be administered in combination with the immunoconjugates of the invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the immunoconjugates of the invention.
  • the immunoconjugates of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685).
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
  • the invention provides protocols for the administration of pharmaceutical composition comprising immunoconjugates of the invention alone or in combination with other therapies to a subject in need thereof.
  • the therapies e.g., prophylactic or therapeutic agents
  • the therapy e.g., prophylactic or therapeutic agents
  • the combination therapies of the present invention can also be cyclically administered.
  • Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and/or to improve, the efficacy of the therapies.
  • a first therapy e.g., a first prophylactic or therapeutic agent
  • a second therapy e.g., a second prophylactic or therapeutic agent
  • the therapies e.g., prophylactic or therapeutic agents
  • the combination therapies of the invention can be administered to a subject concurrently.
  • each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
  • Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route.
  • the therapies are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart.
  • two or more therapies are administered to a within the same patient visit.
  • the prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition.
  • the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions.
  • the prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.
  • grafting of full-length peptide tag grafting of truncated peptide tag
  • insertions both truncated and full-length.
  • grafting of a full-length ybbR tag is exemplified by the mutant anti-hHER2-HC-S132D-K133S-S134L-T135E-S136F-G1371-G138A-T139S-A140K-A141L-L 142 A (SEQ ID NO:102), while the Trastuzumab anti-hHER2-HC-P189G-S190D-S191-S192L-L193S-G194W-T195L (SEQ ID NO:109) mutant constitutes grafting of a truncated S6 tag.
  • mutant anti-hHER2-HC-S190G-S191D-S192-L193-G194S-T195W-Q196L-T197L-RLLN-Y198 (SEQ ID NO:113) wherein residues S190 and S191 were mutated to glycine and aspartic acid, respectively, G194 to serine, T195 to tryptophan, Q196 and T197 to leucine and the truncated S6 tag RLLN (SEQ ID NO:1060) was inserted between L197 and Y198.
  • both truncated and full-length peptide tags were inserted into loops between antibody residues.
  • the peptide-tagged antibodies are named according to the immunoglobulin heavy or light chain, which contains the grafted or inserted peptide tag.
  • the associated unmodified heavy or light chain is not explicitly mentioned.
  • X′ 1 Lys
  • X′ 2 Glu
  • X′ 3 Met
  • X′ 4 Ala
  • the peptide tag was mapped on the selected loops in such a way that the reactive Ser residue was at or near the tip of the loop in order to allow a deeper fit into the active site of Sfp enzyme.
  • the complexes between IgG and Sfp enzyme were constructed next and examined for clashes. Those with significant clashes were rejected and the corresponding loops were excluded from the selection.
  • insertion sites were chosen both by visual inspection of the crystal structure of the human IgG1 B12 antibody (PDB ID: 1 HZH) as well as by calculating the solvent-accessible surface area of residues by using the program ICM from MolSoft LLC.
  • Trastuzumab IgG1 The heavy and light chains of Trastuzumab IgG1 were transiently expressed in mammalian cells using the pOG expression vector under the control of a CMV promoter. Peptide tags for labeling with 4′-phosphopantetheinyl transferases were incorporated into Trastuzumab IgG1 at various positions by standard molecular biology methods. All primers used for cloning are listed in Table 8.
  • HEK293F cells were co-transfected with plasmid DNA encoding the heavy and light chains of Trastuzumab (human kappa isotype).
  • the mammalian cells were cultured in FreeStyleTM 293 Expression Medium at 37° C. under 5% CO 2 , and were split to 0.7 ⁇ 10 6 cells/ml one day prior to transfection. Following transfection, the HEK293F cells were cultured for five days before harvest by centrifugation at 2000 ⁇ g for 30 minutes at 4° C.
  • the resulting medium supernatant was filtered through a 0.22- ⁇ m-pore-size filter.
  • the filtrate was then loaded at a flow rate of about 1 mL/min on a protein A affinity column that was previously equilibrated with 20 column volumes of PBS.
  • the antibody was eluted with 5 column volumes of 0.1 M sodium acetate (pH 3.0).
  • the eluate was immediately neutralized with 10% (v/v) 1 M Tris/HCl (pH 10).
  • Dialysis into PBS was performed using Slide-a-Lyzer dialysis cassettes with 3.5 or 7.0 kDa molecular weight cut-off (Pierce).
  • subtilis Sfp K28E GTCTTTCATTTCACCAGAGGAGCGCGAAAAATGCCGTCGCT 897 AGCGACGGCATTTTTCGCGCTCCTCTGGTGAAATGAAAGAC 898
  • subtilis Sfp T44E AAAGAAGATGCTCACCGCGAGCTGCTGGGAGATGTGCTG 899 CAGCACATCTCCCAGCAGCTCGCGGTGAGCATCTTCTTT 900
  • subtilis Sfp C77Y GCAGGAATATGGCAAACCGTATATTCCAGATCTTCCAGATGC 901 GCATCTGGAAGATCTGGAATATACGGTTTGCCATATTCCTGC 902 E.
  • thermocellum _pET22b/TEV TAAGAAGGAGATATACATATGGGTTTTCTGCCGAAAGAGAAAAAG 994 ACP_ C.
  • the B. subtilis Sfp PPTase was cloned into the pET22b expression vector by using the PIPE method (see Klock et al., Proteins 71:982-994 (2008)). To allow cleavage of the C-terminal His 6 tag (SEQ ID NO:1106), a TEV (tobacco etch virus) protease recognition site was inserted downstream of the Sfp coding sequence. All primers used for cloning are listed in Table 8.
  • Protein expression and purification were performed according to Yin et al. (see Nat. Protoc. 1:280-285 (2006)) with some minor modifications.
  • a 5 mL LB starter culture was inoculated from the glycerol stock of E. coli BL21 (DE3) cells harboring the pET22b/sfp expression plasmid. The culture was grown to saturation by overnight incubation at 37° C. at 300 rpm. The next day, the starter culture was used to inoculate 1 L of TB medium (Sigma), which was agitated at 300 rpm and maintained at 37° C.
  • TB medium Sigma
  • the culture was induced by the addition of IPTG to a final concentration of 1 mM and the temperature was reduced to 30° C.
  • the culture was shaken for another 12-16 hours and the bacterial cells were harvested by centrifugation. Prior to use, the cell pellet was stored at ⁇ 20° C.
  • the frozen pellet was thawed for 15 minutes on ice and re-suspended in a buffer containing 20 mM Tris/HCl (pH 7.9), 0.5 M NaCl, 5 mM imidazole, and 2 U/mL DNase I (3 mL of buffer per g wet weight of cells).
  • Cell lysis was induced by sonication for 4 min, with intervals of 0.5 sec on and 0.5 sec off.
  • the resulting lysate was centrifuged at 40,000 ⁇ g for 20 min at 4° C.
  • His 6 -tagged Sfp enzyme (‘His 6 ’ disclosedas SEQ ID NO: 1106) was then captured by the addition of 4 mL of 50% Ni-NTA slurry (Qiagen) to the cleared lysate. After shaking for 1 hour at 4° C., the resin-lysate mixture was poured into a disposable column (Bio-Rad). The settled resin was washed with 25 column volumes of 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole (pH 8.0) and eluted with 5 column volumes of 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole (pH 8.0).
  • Purified Sfp enzyme was then dialyzed twice against 10 mM Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.5) using a Slide-A-Lyzer Dialysis Cassette (Pierce) with a 3.5 kDa cut-off, and subsequently concentrated to a final concentration of at least 100 ⁇ M using an Amicon Ultra-15 Centrifugal Filter Unit (Millipore) with a 10 kDa cut-off. Finally, the concentrated enzyme was aliquoted, flash-frozen in liquid nitrogen, and stored at ⁇ 80° C.
  • TEV cleavage site was introduced before the C-terminal His 6 tag (SEQ ID NO:1106).
  • Ni-NTA purification of this construct was performed as described above. However, after elution, the Sfp enzyme was exchanged into TEV cleavage buffer containing 50 mM Tris/HCl, 50 mM NaCl (pH 8.0). His 6 tag (SEQ ID NO:1106) removal was carried out by digestion with 7% (w/w) TEV protease at 23° C. for 1 hour and then at 4° C. for 16 hours.
  • the TEV-digested Sfp enzyme was then reloaded onto a Ni-NTA column equilibrated with 1 ⁇ PBS (pH 7.2).
  • the cleaved enzyme was collected from the column flow-through and from a washing step involving 5 column volumes of 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole (pH 8.0).
  • Purified Sfp enzyme was then dialyzed twice against 10 mM Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.5) using a Slide-A-Lyzer Dialysis Cassette (Pierce) with a 3.5 kDa cut-off.
  • Sfp was concentrated to a final concentration of at least 100 ⁇ M using an Amicon Ultra-15 Centrifugal Filter Unit (Millipore) with a 10 kDa cut-off. Finally, the concentrated enzyme was aliquoted, flash-frozen in liquid nitrogen, and stored at ⁇ 80° C.
  • Sfp The purity of Sfp was assessed by SDS-PAGE. His 6 tag (SEQ ID NO:1106) removal was verified by LC-MS and Sfp yield was quantified by ultraviolet spectroscopy at 280 nm (ND-1000 UV-Vis Spectrophotometer, NanoDrop Technologies, Wilmington, Del.) using a molar extinction coefficient of 28620 M ⁇ 1 cm ⁇ 1 . 48 mg of TEV-cleaved Sfp enzyme was obtained per liter culture.
  • TB medium For protein expression, 0.5 L of TB medium was inoculated with a 5 mL starter culture. The culture was agitated at 300 rpm and maintained at 37° C. After reaching an optical density of 0.5 at 600 nm, the culture was induced by the addition of IPTG to a final concentration of 1 mM and the temperature was reduced to 30° C. The culture was shaken for another 16 hours at 300 rpm and the bacterial cells were harvested by centrifugation (15 min at 3400 rpm). Prior to use, the cell pellet was stored at ⁇ 20° C.
  • the frozen pellet was thawed for 10 minutes on ice and re-suspended in a buffer containing 50 mM Tris/HCl (pH 8), 300 mM NaCl, 10 mM imidazole, 1 U/mL DNase I, and CompleteTM EDTA-free protease inhibitor cocktail tablets (Roche) (3 mL of buffer per g wet weight of cells).
  • Cell lysis was induced by sonication for 3 min on ice, with intervals of 0.5 sec on and 0.5 sec off. After incubation for another 10 min on ice, the lysate was centrifuged at 40,000 ⁇ g for 30 min at 4° C.
  • the His 6 -tagged Sfp mutant R4-4 (‘His 6 ’ disclosed as SEQ ID NO: 1106) was then captured by the addition of 2 mL of 50% Ni-NTA slurry (Qiagen) to the cleared lysate. After shaking for 1 hour at 4° C., the resin-lysate mixture was poured into a disposable column (Bio-Rad). The flowthrough was collected and the settled resin was washed with 50 column volumes of 50 mM Tris, 300 mM NaCl, 20 mM imidazole (pH 8.0) and eluted with 5 column volumes of 50 mM Tris, 300 mM NaCl, 250 mM imidazole (pH 8.0).
  • the TEV-digested Sfp mutant R4-4 was then reloaded onto a Ni-NTA column (1 mL bed volume), which was equilibrated with 1 ⁇ PBS (pH 7.2).
  • the cleaved enzyme was collected from the column flow-through and from a washing step involving 5 column volumes of 50 mM Tris, 300 mM NaCl, 20 mM imidazole (pH 8.0).
  • the purified Sfp mutant R4-4 was then buffer-exchanged against 10 mM Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.5) using PD-10 columns.
  • the enzyme had a final concentration of 3.1 mg/mL at a final volume of 17 mL, which corresponds to 105 mg of TEV-cleaved R4-4 mutant per liter culture. Finally, the enzyme was aliquoted into 100 to 1000 ⁇ L fractions, flash-frozen in liquid nitrogen, and stored at ⁇ 80° C. The purity of the enzyme was assessed by SDS-PAGE analysis and His 6 tag (SEQ ID NO:1106) removal was verified by ESI-MS.
  • the AcpS enzyme was expressed in 1 L of TB medium. After shaking the culture at 37° C. with 300 rpm, protein production was induced by the addition of 1 mM IPTG at an optical density of 0.5 (600 nm). Protein expression was carried out overnight at 30° C. and 300 rpm. The next day, the cells were harvested by centrifugation at 3400 rpm for 15 min. The cell pellet was stored at ⁇ 20° C. prior to protein purification.
  • the frozen pellet was thawed for 10 min on ice and resuspended in buffer (3 mL of buffer per g wet weight of cells) containing 50 mM Tris/HCl (pH 8), 300 mM NaCl, 10 mM imidazole, 1 U/mL DNase I, and CompleteTM EDTA-free protease inhibitor cocktail tablets (Roche).
  • Cell lysis was achieved by sonicating the cell suspension on ice for 3 min with intervals of 0.5 sec on and 0.5 sec off. After another incubation period of 10 min on ice, the lysate was centrifuged at 40,000 g for 30 min at 4° C.
  • Ni-NTA slurry was added to the cleared lysate and the lysate/resin mixture was shaken for 1 hour at 4° C.
  • the lysate/resin mixture was poured into a disposable column.
  • the Ni-NTA column was washed with 50 column volumes of buffer containing 50 mM Tris (pH 8), 300 mM NaCl, and 20 mM imidazole. Elution was performed with 5 column volumes of buffer containing 50 mM Tris (pH 8), 300 mM NaCl, and 250 mM imidazole.
  • T. maritima PPTase expression was carried out at a 1 L scale in native FM medium by inoculation with a 10 mL saturated starter culture. The 1 L culture was shaken at 300 rpm at a temperature of 37° C. After 2.5 hours, the culture reached an optical density of 0.5 at 600 nm. Protein production was induced by the addition of arabinose to a final concentration of 0.1% (w/v) and the culture was shaken for an additional 4 hours. Cells were harvested by centrifugation at 4000 rpm for 15 minutes and the cell pellets were stored at ⁇ 20° C. Initial purification of T.
  • maritima PPTase was performed by IMAC (immobilized metal affinity chromatography) using Ni-NTA agarose resin (Qiagen). Cell pellets were thawed and resuspended in 60 mL lysis buffer (40 mM Tris buffer (pH 8.0), 300 mM NaCl, 10 mM Imidazole, 1 mM TCEP). The cell suspension was sonicated on ice for 1.5 minutes (using 1 sec pulses) and centrifuged at 15000 rpm for 30 minutes at 5° C. The cleared lysate was loaded onto a 1.5 mL Ni-NTA column.
  • IMAC immobilized metal affinity chromatography
  • wash buffer 40 mM Tris buffer (pH 8.0), 300 mM NaCl, 40 mM imidazole, 10% glycerol, 1 mM TCEP.
  • Protein elution was carried out with 2 column volumes of elution buffer (20 mM Tris buffer (pH 8.0), 150 mM NaCl, 300 mM Imidazole, 1 mM TCEP).
  • Ni-NTA eluate was further purified using a Superdex 75 column (GE Healthcare) connected to an Akta FPLC system. Size-exclusion chromatography (SEC) was performed at flow rate of 1 mL/min in 10 mM Tris buffer (pH 7.4) supplemented with 1 mM EDTA and 10% (v/v) glycerol. After analyzing protein-containing fractions by SDS-PAGE, fractions containing the T. maritima PPTase were pooled and dialyzed again against the buffer previously used for SEC. The purified enzyme was then concentrated using an Amicon Ultra-15 Centrifugal Filter Unit (Millipore) with a 10 kDa cut-off.
  • SEC Size-exclusion chromatography
  • Precipitate was removed by centrifugation at 13000 rpm for 2 min using a table top centrifuge.
  • the concentrated protein (1.0 mg/mL, 48 ⁇ M) was aliquoted into 100 ⁇ L fractions, flash-frozen in liquid nitrogen, and stored at minus 80° C.
  • the purity of T. maritima PPTase was assessed by SDS-PAGE and the yield was quantified by Bradford assay using BSA as standard. After all purification steps, 1.4 mg of AcpS enzyme was obtained per liter culture.
  • Tetramethylrhodamine-C2-maleimide (5.5 mg, 10.4 mmol) dissolved in 300 ⁇ L of DMSO was added to CoA (10.4 ⁇ mol in 150 ⁇ L water) in 750 ⁇ L of 10 ⁇ PBS buffer and stirred at 23° C. for 1 hour. After the reaction, the reaction mixture was lyophilized to obtain the crude product, which was purified by RP-C18 flash chromatography. Fractions of the desired product were combined and lyophilized to afford CoA-maleimidoethylamido-tetramethylrhodamine (9.8 mg with 94.4% purity) as a dark purple powder.
  • ESI-MS calculated for C 52 H 64 N 11 O 22 P 3 S [MH] + : 1320.3; observed: 1320.3.
  • MC-MMAF (see Doronina et al., Bioconj. Chem. 17:114-124 (2006)) (36.0 mg, 38.9 ⁇ mol) dissolved in 1.8 mL of DMSO was added to CoA (39.0 ⁇ mol in 312 ⁇ L water) in 2.9 mL of 10 ⁇ PBS buffer and stirred at 23° C. for 1 hour. After the reaction, the reaction mixture was lyophilized to obtain the crude material, which was purified by RP-C18 flash chromatography. Fractions of the desired product were combined and lyophilized to afford CoA-MC-MMAF (35.5 mg with 97.5% purity) as a white powder. ESI-MS calculated for C 70 H 112 N 13 O 27 P 3 S [MH] + : 1691.7; observed: 1691.2.
  • Sfp enzyme was added to give a final concentration of typically 1 ⁇ M.
  • the enzymatic reaction was allowed to proceed at either 23° C. or 37° C. for 16 hours. After this time period, the reaction progress was analyzed by ESI-MS and HPLC.
  • anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3 SEQ ID NO:94, FIG. 5D
  • anti-hHER2-HC-E388-DSLEFIASKL-N389 SEQ ID NO:130, FIG. 5E
  • anti-hHER2-HC-E388-DSLEFIASKLA-N389 SEQ ID NO: 129, FIG. 5F
  • mAb2-HC-T359-GDSLSWLLRLLN-K360 SEQ ID NO:148, FIG. 5G
  • DAR drug-to-antibody-ratio
  • ESI-MS of reduced conjugate samples suggest site-specific modification of only the heavy chain as designed.
  • the trastuzumab immunoconjugates (A) anti-hHER2-HC-V2-GDS-ppan-MC-MMAF-LSWLLRLLN-Q3 (SEQ ID NO:1120), (B) anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389 (SEQ ID NO:1122), and (C) anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKL-N389 (SEQ ID NO:1121) were analyzed by analytical size-exclusion chromatography (AnSEC) on a Shodex PROTEIN KW-803 column. In all three cases, the ADCs were monomeric (no detectable amounts of aggregated material).
  • This example represents two Sfp-catalyzed conjugations of CoA-tetramethylrhodamine (CoA-TMR) to Trastuzumab antibodies with either grafted or inserted S6 tags performed as described in Example 6.
  • HPLC traces of reaction mixtures were monitored at both 280 nm and 555 nm ( FIG. 9 ). The latter wavelength is near the absorption maximum of the TMR dye ( ⁇ 550 nm).
  • maleimide-linked payloads may undergo deconjugation in plasma via maleimide exchange with reactive thiols of albumin, glutathione, and cysteine (Alley et al., Bioconjugate Chem. 2008, 19, 759-765).
  • Maleimide-based conjugates can be stabilized through chemical ring-opening of the maleimidocaproyl linkage (see, Shen et al., Nature Biotech. 30:184-189 (2012)).
  • the respective ADC of anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 was prepared using CoA-open-ring-MC-MMAF.
  • the HPLC results indicate that the pH range 8 to 9 is optimal for the conjugation of CoA-MC-MMAF to peptide-tagged Trastuzumab.
  • plotting the percentage of ADC with a DAR of 2 against the pH indicates that the pH optimum is independent of the insertion site of the S6 tag for the two sites tested.
  • DSF differential scanning fluorometry
  • DSC differential scanning calorimetry
  • unmodified trastuzumab exhibits two transitions. The transitions were observed at 69.7 and 81.1 degrees Celsius by DSF and 72.3 and 81.0 degrees Celsius by DSC. Similar to the unmodified antibody, most CoA-MC-MMAF immunoconjugates exhibit two transitions although with different amplitudes ( FIG. 15 ). DSF and DSC measurements of thermal melting points agree well although DSF reports a roughly 2 degree lower first transition. Generally, most engineered, non-conjugated antibodies and the respective peptide-tagged ADCs show little destabilization as compared to the wild-type antibody anti-hHER2.
  • mice To check the in vivo stability of two peptide-tagged Trastuzumab ADCs with MMAF payload (DAR of 2), we conducted a pharmacokinetic (PK) study in mice.
  • Anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360 (SEQ ID NO:1117) and anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 (SEQ ID NO:1118) were injected i.v. into 3 mice using ADC concentrations of 1.0 mg/kg. 10 samples were collected at 0.2, 1, 3, 7, 24, 48, 96, 168, 240, and 336 hours.
  • the plasma titers of both ADCs were monitored up to two weeks using ELISA assays with anti-human IgG as well as anti-MMAF antibodies and ELISA plates coated with truncated human HER2 (extracellular domains 3-4). The ELISA results were then compared to PK studies of an unmodified Trastuzumab IgG1.
  • anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360 showed a fast decay in mice in comparison to unmodified trastuzumab
  • anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 SEQ ID NO:1118
  • exhibited a serum clearance similar to unmodified trastuzumab over a two week time period FIG. 16 .
  • anti-hIgG and anti-MMAF titers track each other, suggesting that little if any drug is lost during the in vivo exposure in mice.
  • the bioorthogonality of PPTase-catalyzed 4′-phosphopantetheinylation enables the site-specific labeling of peptide-tagged IgGs in complex mixtures such as conditioned medium.
  • exogenously added PPTase such as Sfp
  • drug-CoA substrate such as CoA-MC-MMAF
  • HEK293F cells were transfected with plasmid DNA coding for IgG1 heavy chain with S6 tag insertion in the CH3 domain and plasmid DNA coding for unmodified kappa light chain.
  • the 40 mL HEK293F suspension culture was cultured for five days at 37° C. After harvesting by centrifugation at 2000 rpm for 10 minutes, the medium supernatant was supplemented to a final concentration of 40 ⁇ M of CoA-MC-MMAF, 10 mM of MgCl 2 , and 50 mM of HEPES (pH 7.5). The medium supernatant was then split into two 20 mL aliquots. Recombinantly produced Sfp enzyme (5 ⁇ M) was added to one of the aliquots (see Table 20, Experiment #2) and the enzymatic reaction was allowed to proceed for 24 hours at room temperature.
  • Antibody purification was carried out using Protein A Sepharose Fast Flow columns with 0.25 mL bed volume for each experiment. After equilibration with PBS, the medium supernatants were applied to the columns at a flow rate of about 1 mL/min and the flowthrough was collected. Following washing with 20 column volumes of PBS, bound antibody was eluted using 6 column volumes of 0.1 M sodium acetate (pH 3.0) followed by immediate neutralization with 1 M Tris/HCl (pH 10) to reach a final pH of about 8. The purity of the eluates was assessed by SDS-PAGE analysis and the antibody yield was determined by the Bradford method. Finally, Sfp-dependent in-medium ADC formation was confirmed by ESI-MS and HPLC analysis of the Protein A eluates.
  • the principle of the preparation of immune conjugates via acetyl CoA is a three-step chemoenzymatic conjugation protocol in which the acetyl moiety serves as a protecting group for the reactive thiol group of CoA.
  • PPTases such as Sfp tolerate large CoA analogues (e.g. peptidyl-CoA) for catalysis
  • the catalytic efficiency (k cat /K M ) is significantly reduced compared to CoA itself (see, Sieber et al., J. Am. Chem. Soc. 125: 10862-10866 (2003)).
  • the small acetyl group ensures similar enzyme kinetics as seen for the native CoA substrate.
  • IgG antibody is carried out as described in Example 6 using acetyl CoA instead of CoA-MC-MMAF.
  • the conjugate is dialyzed into Reaction Buffer (0.1 M sodium phosphate (pH 7.2), 0.15 M NaCl).
  • the dialyzed conjugate is concentrated to about 5 mg/mL and supplemented with 10% (v/v) of Deacetylation Solution containing Reaction Buffer (pH 7.2) with 0.5 M hydroxylamine and 25 mM EDTA.
  • This chemical thioester cleavage reaction is allowed to proceed for 3 hours at room temperature, followed by buffer-exchanging the reaction mixture into Reaction Buffer (pH 7.2) supplemented with 10 mM EDTA. After confirmation of quantitative deacetylation by ESI-MS, the deprotected ppan moiety is then conjugated with 15 equivalents of thiol-reactive maleimide-MC-MMAF (0.5 mM) for 1 hour at room temperature. The reaction is quenched by buffer-exchange into PBS. Finally, quantitative ADC formation is confirmed by ESI-MS and HPLC analysis.
  • the bioorthogonality of PPTase-catalyzed generation of homogeneous ADCs allows the site-specific labeling of IgGs in cell culture media (see Example 19). Instead of directly attaching the cytotoxic drug molecule to the antibody, it is also possible to carry out in-medium labeling with acetyl CoA for ADC generation via a three-step chemoenzymatic conjugation process.
  • the small acetyl CoA analogue allows conjugation reactions with improved catalytic efficiency (k cat /K M ) as compared to large cytotoxic CoA analogues, thereby significantly decreasing the amount of enzyme needed for quantitative conjugation. Furthermore, for process development, it would be advantageous to perform labeling reactions in large culture volumes with non-toxic compounds.
  • the peptide-tagged IgG conjugated with the acetyl-ppan moiety can be purified in a single step using protein A affinity chromatography.
  • the two subsequent chemical reactions are carried out as described in Example 20.
  • the bioorthogonality of PPTase-catalyzed generation of homogeneous ADCs also allows the site-specific labeling of IgGs in cell culture media (see Example 19). Instead of directly attaching the cytotoxic drug molecule to the antibody, it is also possible to carry out in-medium labeling with acetyl CoA for ADC generation via a three-step chemoenzymatic conjugation process.
  • the small acetyl CoA analogue allows conjugation reactions with improved catalytic efficiency (k cat /K M ) as compared to large cytotoxic CoA analogues, thereby significantly decreasing the amount of enzyme needed for quantitative conjugation. Furthermore, for process development, it would be advantageous to perform labeling reactions in large culture volumes with non-toxic compounds.
  • the peptide-tagged IgG conjugated with the acetyl-ppan moiety can be purified in a single step using protein A affinity chromatography.
  • the two subsequent chemical reactions are carried out as described in Example 20.
  • in-medium labeling can also be performed with CoA analogues covalently linked to bioorthogonal groups, such as azido, alkene, alkyne, ketone, or aldehyde moieties.
  • bioorthogonal groups such as azido, alkene, alkyne, ketone, or aldehyde moieties.
  • Example 22 exemplifies the two-step method for the site-specific attachment of carbonyl-functionalized CoA analogues to an A1-tagged antibody followed by oxime ligation of the terminal group (TG).
  • TG terminal group
  • the resulting carbonyl-functionalized antibody is purified by protein A affinity purification.
  • the second step then involves reaction of the ppan-linked carbonyl group with an aminooxy-derivatized TG. Following reaction, excess TG is removed by dialysis or buffer exchange.
  • the synthesis of a carbonyl-functionalized CoA analogue (ketone CoA) is described in Example 23.
  • ADCs with a DAR of 4 can be generated by inserting/grafting multiple peptide tags into an antibody, which are substrates of the same enzyme ( FIG. 19A ).
  • both the ybbR- and the S6-tags are recognized as substrates by the PPTase Sfp.
  • labeling of antibodies with multiple different ligands is achieved by inserting/grafting peptide tags into an antibody, which are substrates of two different PPTases.
  • the A1 tag is exclusively recognized by the AcpS PPTase, while the S6 tag is preferentially modified by the Sfp PPTase.
  • immunoconjugates with higher DARs may be generated by adding additional tags.
  • Enzymatic conjugation can also be combined with other labeling strategies such as site-specific conjugation through cysteine, pyrrolysine, pyrroline-carboxy-lysine, and unnatural amino acids as well as chemoselective methods such as Lys, Cys or Tyr selective chemistries.
  • the resulting medium supernatant was filtered through a 0.22- ⁇ m-pore-size filter. Purification was accomplished using a Protein A Sepharose Fast Flow column (GE Healthcare) with a bed volume of 0.6 mL, which was equilibrated with 20 column volumes of PBS. The filtered medium supernatant was loaded at a flow rate of about 1 mL/min. After washing the column with 20 column volumes of PBS, the peptide-tagged antibody was eluted with 5 column volumes of 0.1 M sodium acetate (pH 3.0) followed by immediate neutralization with 1 M Tris/HCl (pH 10) to a final pH of about 8. According to the Bradford method, the total yield was 8 mg of purified antibody per liter culture.
  • a Protein A Sepharose Fast Flow column GE Healthcare
  • the filtered medium supernatant was loaded at a flow rate of about 1 mL/min.
  • the peptide-tagged antibody was eluted with 5 column volumes of 0.1 M sodium acetate (
  • the purity of the antibody construct was assessed by SDS-gel electrophoresis. After concentration with a 30 kDa cut-off Amicon Ultra Centrifugal Filter Unit, 2.5 ⁇ M anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388-DSLEFIASKLA-N389 (SEQ ID NO:142) was incubated with 50 ⁇ M CoA-MC-MMAF, 1 ⁇ M Sfp, 12.5 mM MgCl 2 , in 75 mM HEPES buffer, pH 7.5, at 23° C. for 16 hours to enzymatically label the dual-tagged antibody with four drug molecules.
  • the deconvoluted mass spectrum of the reduced and deglycosylated antibody construct confirmed the covalent attachment of two ppan-MC-MMAF units to each heavy chain of Trastuzumab (observed mass, 54223.20 Da; expected mass, 54231 Da). Neither uncoupled (expected mass, 51700 Da) nor mono-labeled species (expected mass, 52966 Da) were observed by ESI-MS. Near quantitative conversion to an ADC with a DAR of 4 (95% according to peak area integration) was further confirmed by HPLC analysis ( FIG. 19B ).
  • each filtrate was loaded onto a Protein A affinity column containing 0.2 mL of settled resin at an approximate flow rate of 1 mL/min. The column was then washed with 20 column volumes of PBS followed by elution with 5 column volumes of 0.1 M sodium acetate, pH 3.0. The eluate was immediately neutralized with 25% (v/v) of 1 M Tris-HCl (pH 8.0).
  • Protein A-purified antibodies To determine the yield of the Protein A-purified antibodies (Table 21), protein concentrations of the eluates were measured in duplicate on a ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies) at 280 nm using the preset molar extinction coefficient for IgG molecules. After concentrating the peptide-tagged antibodies using 30 kDa cut-off Amicon Ultra-0.5 centrifugal filter devices (EMD Millipore), enzyme-catalyzed conjugation reactions were performed for about 16 hours at 20° C.
  • the peptide-tagged ADC constructs were further purified by Ni-NTA (nickel-nitrilotriacetic acid) chromatography to remove Sfp enzyme and excess CoA-MC-MMAF substrate.
  • Ni-NTA nickel-nitrilotriacetic acid
  • the concentrated conjugation samples were loaded onto the columns at an approximate flow rate of 1 mL/min.
  • the columns were washed with 20 column volumes of PBS followed by elution with 5 column volumes of Tris-HCl buffer (50 mM, pH 8.0) supplemented with 250 mM imidazole and 300 mM NaCl.
  • the recovery of the peptide-tagged ADCs averaged 39% of the Protein A-purified starting material.
  • the PEPP system was then used to buffer-exchange each sample into PBS using NAP-10 Columns (GE Healthcare).
  • the peptide-tagged ADCs were concentrated using Amicon Ultra-4 centrifugal filter devices (EMD Millipore), and the concentrations of the conjugates were adjusted by dilution with PBS. Adjusted to the appropriate concentration, the ADC samples were further characterized by DSF (differential scanning fluorimetry), LC90 (LabChip 90), AnSEC (analytical size-exclusion chromatography), and in vitro potency assays (data not shown).
  • the expression levels also depend on the type of peptide tag: 95 antibody constructs with ybbR tag insertions on average show higher expression levels (19 ⁇ 7 mg per liter culture) than the corresponding 88 constructs with S6 tag insertions (13 ⁇ 8 mg per liter culture).
  • the opposite trend is observed for the conjugation efficiencies based on reverse-phase HPLC analysis: 44 (72%) peptide-tagged constructs with near quantitative ADC formation (drug-to-antibody ratio 1.9) are based on insertion of the S6 peptide sequence, while only 17 (28%) ybbR-tagged antibodies displayed near quantitative conversion to the corresponding ADC.
  • Thermostability of the resulting ADCs depends on the site of peptide tag insertion. For instance, most peptide tag insertions in the CH2 domain lead to a significant decrease of the lowest observed thermal transition (Tm1) according to DSF (differential scanning fluorimetry) measurements as will be illustrated in more detail in Example 26. Little aggregation or antibody oligomers were observed for 135 (87%) out of 156 peptide-tagged ADCs that were examined by analytical size-exclusion chromatography 90% monomeric species). The in vitro potency of the peptide-tagged ADCs correlated as expected with their degree of labeling. Although a large number of peptide-tagged ADCs with preferred properties can be generated, the data also illustrate that expression yield, thermal stability, conjugation efficiency and other properties are greatly affected by the choice of tag insertion site.
  • the antibody constructs were eluted with 0.1 M sodium acetate (pH 3.0) in two 2.5 mL fractions. Both fractions were immediately neutralized with 25-38% (v/v) of Tris-HCl buffer (1 M, pH 8.0).
  • Tris-HCl buffer 1 M, pH 8.0.
  • protein concentrations of the eluates were measured in duplicate on a ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies) at 280 nm according to the preset molar extinction coefficient for IgG molecules.
  • the enzymatic reaction was allowed to proceed for about 16-20 hours at room temperature, before verifying the degree of labeling by analytical reverse-phase HPLC using the respective uncoupled antibody as control (Table 22). All conjugation reactions were analyzed by mass spectrometry on an Agilent 6520 Q-TOF instrument (Table 22). After confirming near quantitative conjugation, reaction mixtures were concentrated to a final volume of 1 mL using 30 kDa cut-off Amicon Ultra centrifugal filter devices (EMD Millipore).
  • b Yield of antibody per liter culture (based on 200-1000 mL cultures) measured after protein A purification.
  • c Yield of ADC per liter of culture measured after size-exclusion chromatography.
  • d Drug-to-antibody ratio according to HPLC.
  • e Analytical size exclusion chromatography results for ADC (percent of monomer).
  • f Mass in Dalton as predicted for the ADC.
  • h Observed mass corresponds to non-conjugated antibody.
  • i Observed mass corresponds to non-clipped C-terminal lysine residue of heavy chain.
  • j Observed mass presumably corresponds to sodium adduct.
  • k Observed mass corresponds to clipped Gly446 residue of heavy chain.
  • l Observed mass corresponds to an unknown species of low abundance.
  • j Carryover peak n.d., not determined.
  • Expression levels of the selected peptide-tagged antibodies averaged 25 mg per liter of cell culture (ranging from 4 to 57 mg/L) (Table 22) and the final yield of purified ADC averaged 14 mg per liter of cell culture (ranging from 1 to 38 mg/L) (Table 22).
  • All ADCs were site-specifically conjugated with two CoA-MC-MMAF molecules at an average DAR of 1.9 (DARs ranging from 1.5 to 2.0) as verified by HPLC and MS (Table 22). No aggregation or oligomeric species were detected for 92 of 97 ADCs prepared (Table 22). All other ADCs were at least 81% monomeric as determined by analytical size exclusion chromatography (no data for two ADCs).
  • the thermal stability was significantly (>3 degree C.) reduced relative to wild-type trastuzumab as illustrated by the difference in Tm1.
  • This transition is attributed to the unfolding of the CH2 domain (amino acid residues 228-340) of an IgG and indeed most of the antibodies that are destabilized have the peptide-tag inserted at positions in the CH2 domain.
  • the plot of FIG. 18 illustrates that peptide tag insertions into the CH2 domain generally lead to lower Tm1 values than those of respective peptide tag insertions into the adjacent CH1 and CH3 domains of the heavy chain.
  • the location of the peptide tag can significantly affect the properties of the resulting antibody and ADC.
  • Purified ADCs were further characterized for in vitro potency against selected cell lines (Table 24) including two engineered cell lines, MDA-MB231 clone 16 and clone 40, and two cell lines (JimT1 and HCC1954) that endogenously express the targeted antigen, human HER2, on the cell surface.
  • MDA-MB231 clone 16 cells stably express 500,000 copies of HER2 per cell while clone 40 expresses only 5000 copies/cell.
  • HCC1954 cells endogenously express high level (500,000 copies/cell) of human HER2 on the surface (Clinchy B, Gazdar A, Rabinovsky R, Yefenof E, Gordon B, Vitetta E S. Breast Cancer Res Treat.
  • the JimT1 cell line expresses approximately 80,000 copies of HER2 per cell (Mocanu M-M, Fazekas Z, Petras M, Nagy P, Sebestyen Z, Isola J, Timar J, Park J W, Vereb G, Szollosi J. Cancer Letters (2005) 227: 201-212).
  • the cell proliferation assays were conducted with Cell-Titer-GloTM (Promega) five days after cells were incubated with various concentrations of ADCs (Riss et al., (2004) Assay Drug Dev Technol. 2:51-62) with an automated system (Melnick et al., (2006) Proc Natl Acad Sci USA.
  • trastuzumab peptide-tagged-MMAF ADCs specifically killed MDA-MB231 clone 16, HCC1954 and JimT1 cells (Table 24): IC 50 values of the trastuzumab peptide-tagged-MMAF ADCs averaged around 0.45 nM, 0.24 nM and 2.0 nM for MDA-MB231 clone 16, HCC1954 and JimT1 cells, respectively (Table 24), consistent with the different HER2 expression levels. No killing of the antigen negative (Her2 low) control cell line MDA-MB231 clone 40 was observed at the highest test concentration (33 nM) for 92 of 97 ADCs.
  • Each peptide-tagged MMAF ADC was injected intravenously into three mice at a single dose of 1 mg/kg.
  • Nine plasma samples were then collected over a time course of 340 hours before plasma titers of the ADCs were determined by ELISA.
  • the ELISA assay uses the immobilized extracellular domain of human HER2 for capturing trastuzumab ADC molecules from plasma samples.
  • an anti-MMAF antibody is used to exclusively measure the plasma concentration of the “intact” trastuzumab MMAF conjugate.
  • an anti-hIgG antibody generates a signal indicating the plasma concentration of both conjugated and unconjugated trastuzumab molecules.
  • both anti-MMAF and anti-hIgG ELISAs are expected to provide identical readouts on ADC plasma concentration.
  • the anti-MMAF ELISA is expected to produce a lower signal than the anti-hIgG ELISA.
  • the comparison of both ELISA signals therefore allows the quantification of payload deconjugation during the in vivo exposure of the respective ADC.
  • the interpretation of the PK data is based on standard curves that were generated with the same ADCs as used for intravenous injection into mice.
  • AUC area-under-the-plasma-concentration-versus-time-curve
  • FIG. 20 A-C exemplifies PK curves of three peptide-tagged MMAF ADCs displaying high AUC hIgG values (ADC of SEQ ID NO:248, 28334 nM*hr; ADC of SEQ ID NO:33, 21011 nM*hr; ADC of SEQ ID NO:251, 21689 nM*hr).
  • PK curves of three constructs showing low AUC hIgG values are illustrated in FIG. 20 D-F.
  • ADC of SEQ ID NO:218, 1362 nM*hr; ADC of SEQ ID NO:202, 1757 nM*hr; ADC of SEQ ID NO:244, 2378 nM*hr are illustrated in FIG. 20 D-F.
  • AUC hIgG values both anti-hIgG and anti-MMAF titers track each other, suggesting that little if any payload deconjugation occurred in vivo.
  • the rapid clearance observed for some of the peptide-tagged ADCs is likely the result of inserting an S6, A1 or ybbR peptide sequence into specific regions of the IgG1 molecule rather than drug attachment.
  • the putative relationship between tag insertion site and pharmacokinetic profile is exemplified by the two peptide-tagged MMAF ADCs of SEQ ID NO:218 and SEQ ID NO:202, which display the lowest and third lowest measured AUC hIgG values of 1362 nM*hr and 1757 nM*hr, respectively. Both ADCs contain S6 tag insertions in the CH2 domain of the heavy chain.
  • these ADCs In addition to the instability in murine circulation, these ADCs also exhibit the fifth lowest and ninth lowest thermostabilities of the 86 tested samples of the PK study. According to DSF measurements, the corresponding ADCs display Tm1s of 49.0° C. (ADC of SEQ ID NO:218) and 51.2° C. (ADC of SEQ ID NO:202), resulting in a decrease of 20.7° C. and 18.5° C., respectively, in comparison to wild-type trastuzumab having a Tm1 of 69.7° C. In contrast, the forty ADCs with the highest AUC hIgG values (19695-32553 nM*hr) display an average Tm1 value of 67.4° C., which is only 2.3° C.
  • These include antibodies with heavy chain insertions between S119-T120, T120-K121, T135-S136, S136-G137, G138-T139, A162-L 163 , T164-S165, S165-G166, G194-T195, T195-Q196, and E388-N389 (CH3 domain) corresponding to SEQ ID numbers 126, 127, 129, 130, 131, 132, 149, 151, 152, 157, 158, 160, 166, 168, 169, 178, 179, 250, 251, 256, 257, 259, 265, 267, 268, 277, 278, 356, 358, 359, 364, 365, 367, 371, 373, 374, 380, and 381.
  • the gene encoding the AcpS PPTase was cloned into the mammalian expression vector pRS, which appends the N-terminal signal sequence MKTFILLLWVLLLWVIFLLPGATA (SEQ ID NO:355).
  • the construct, pRS-AcpS also adds a C-terminal His 6 tag to the recombinant enzyme (SEQ ID NO: 1106).
  • an oligonucleotide fragment encoding the 12-amino-acid A1 peptide sequence was inserted into the heavy chain gene of the antibody mAb2-HC (SEQ ID NO:147) in the mammalian expression vector pM4, resulting in the construct pM4-A1.
  • This plasmid also co-expresses the corresponding light chain under the CMV promoter.
  • 293 FreestyleTM cells were transiently transfected with a 1:1 mixture of the recombinant expression plasmids pM4-A1 and pRS-AcpS, and cultured in five aliquots of 200 mL of FreestyleTM expression media (Invitrogen) for five days at 37° C. under 5% CO 2 .
  • the cell cultures were harvested by centrifugation at 2,000 rpm for 10 min, passed through 0.22 ⁇ m filters, and pooled.
  • the labeling reactions were then initiated by addition of acetyl CoA substrate (Sigma-Aldrich) to a final concentration of 1 mM.
  • acetyl CoA substrate Sigma-Aldrich
  • the resulting reaction mixtures with volumes of 1.5 mL to 15 mL were incubated for approximately 16 h at 37° C.
  • reaction mixtures were purified by Ni-NTA and Protein A affinity chromatography, respectively. With the exception of the unconcentrated sample, all reaction mixtures were diluted two-fold with PBS prior to loading onto PBS-equilibrated Protein A-Sepharose columns (0.5 mL bed volume, GE Healthcare) at an approximate flowrate of 1 mL/min. The column flowthrough was directly applied to PBS-equilibrated IMAC columns filled with 0.5 mL of Ni-NTA Agarose (Qiagen).
  • the A1-tagged antibody was eluted from the Protein A affinity columns with 6 column volumes of 0.1 M sodium acetate buffer (pH 3.0) followed by immediate neutralization with 12% (v/v) of 1 M of Tris-HCl buffer (pH 10).
  • the purified antibody constructs were concentrated using 30 kDa cut-off Amicon Ultra centrifugal filter units, reduced, and deglycosylated followed by mass spectrometric analysis on an Agilent 6520 Q-TOF instrument (Agilent Technologies). As shown in Table 26, a two-fold concentration factor of the conditioned cell-culture medium is sufficient for near quantitative conjugation in the presence of 1 mM of acetyl CoA substrate, 0.16 ⁇ M (24 mg/L) of A1-tagged antibody, and 1.1 ⁇ M (17 mg/L) of AcpS enzyme.
  • the acetyl group of the acetyl CoA substrate is completely cleaved off during in-medium labeling, thereby indicating hydrolysis of the thioester bond in conditioned cell-culture medium.
  • this negative control therefore excludes the presence of significant amounts of CoA or one of its analogues in the cell-culture medium.
  • the experiment demonstrates that a peptide-tagged antibody can be quantitatively labeled with a supplemented CoA analogue in 2-fold concentrated cell-culture medium via PPTase catalysis. Because antibody concentrations during fermentation of production cell lines is significantly higher than in the current proof-of-concept experiments, it can be anticipated that enzymatic conjugation of supplemented CoA analogues to a peptide-tagged antibody will be scalable to production levels.
  • the supplemented CoA analogues will feature a thiol group, a protected thiol group or a bioorthogonal reactive group such as an aldehyde, a keto group, an azido or an alkyne group.
  • the antibody enzymatically activated with a reactive group could be reacted with a complementary toxin analogue to afford the corresponding ADC in the second step.
  • the goal of this experiment is to demonstrate the feasibility to site-specifically attach a bioorthogonal group to a peptide-tagged antibody in conditioned cell-culture medium.
  • the first step of the two-step method was carried out with the ketone CoA analogue whose synthesis has been described in Example 23.
  • Successful in-medium labeling of a peptide-tagged antibody with this carbonyl-functionalized CoA analogue will allow subsequent attachment of an aminooxy-functionalized payload via oxime ligation in the second step of the two-step method (see also FIG. 22 ).
  • an aliquot of 60 mL of cleared cell-culture medium was concentrated 20-fold using 30 kDa cut-off Amicon Ultra centrifugal filter units (EMD Millipore). After removing precipitate by centrifugation at 3,724 ⁇ g for 5 min, the labeling reaction was initiated by supplementing 1.31 mL of concentrate with ketone CoA at a final concentration of 1 mM and 10-fold reaction buffer (pH 8.8) at a final concentration of 75 mM of Tris-HCl and 10 mM of MgCl 2 . The enzymatic reaction in a total volume of 1.5 mL was incubated for approximately 16 h at 37° C.
  • the reaction mixture Prior to analyzing the degree of labeling with carbonyl-functionalized CoA analogue by mass spectrometry, the reaction mixture was purified by protein A affinity chromatography. After two-fold dilution with PBS, the diluted reaction mixture was loaded onto a PBS-equilibrated Protein A-Sepharose column (0.6 mL bed volume, GE Healthcare) at an approximate flowrate of 1 mL/min. The column matrix was washed with approximately 40 bed volumes of PBS before the retained material was eluted with 6 column volumes of 0.1 M sodium acetate buffer (pH 3). Finally, the eluate was neutralized by addition of 12% (v/v) of 1 M of Tris-HCl buffer (pH 10).
  • the purity of the antibody was assessed by reducing SDS-PAGE. According to UV-Vis measurements on a NanoDrop ND-1000 Spectrophotometer, 0.34 mg of antibody was recovered, corresponding to an antibody concentration of 1.5 ⁇ M (230 mg/L) in the 20-fold concentrated cell-culture medium. This exactly reproduces the measured antibody concentration during the labeling reaction with acetyl CoA in 20-fold concentrated cell-culture medium (Example 27). To assess the degree of antibody labeling with ketone CoA by mass spectrometry, the neutralized eluate was concentrated using 30 kDa cut-off Amicon Ultra centrifugal filter units, deglycosylated, and reduced.
  • Mass spectrometric analysis on an Agilent 6520 Q-TOF instrument indicated formation of the desired carbonyl-functionalized antibody conjugate (observed, 51995.32; expected, 51999.6), with formation of about 24% of 4′-phospho-pantetheine-modified antibody as a side product (observed, 51925.42; expected, 51929.6). No unconjugated antibody was detectable in the deconvoluted mass spectrum (expected, 51589.2). The presence of 4′-phosphopantetheine-modified antibody as a side product might be explained by the incomplete formation of ketone CoA during the reaction between CoA-SH and methyl vinyl ketone (Example 23).
  • the in vivo efficacy of the ybbR-tagged trastuzumab ADC anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389 was assessed by using a xenograft tumor model, which is based on the implantation of a human tumor cell line into immune-deficient nude mice. As described previously (Sausville and Burger, 2006), studies with such tumor xenograft mice have provided valuable insights into the in vivo efficacy of anti-cancer reagents.
  • the in vivo efficacy study was carried out with nu/nu mice that were subcutaneously injected with MDA-MB231 clone 16 cells (Morton and Houghton, 2007).
  • This cell line was chosen based on previous in vitro potency assays revealing its high sensitivity to the aforementioned ybbR-tagged MMAF ADC in an antigen dependent manner (see Table 24).
  • the ybbR-tagged MMAF ADC was intravenously injected in a single dose at either 5 mg/kg or 3 mg/kg, with each treatment group comprising nine mice. After administering the antibody-drug conjugate, the tumor growth was monitored weekly. As shown in FIG.
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US9585970B2 (en) 2017-03-07
US20170218085A1 (en) 2017-08-03
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KR20150027072A (ko) 2015-03-11
BR112014030098A2 (pt) 2017-07-25
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