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US20070122406A1 - Optimized proteins that target Ep-CAM - Google Patents

Optimized proteins that target Ep-CAM Download PDF

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US20070122406A1
US20070122406A1 US11/484,198 US48419806A US2007122406A1 US 20070122406 A1 US20070122406 A1 US 20070122406A1 US 48419806 A US48419806 A US 48419806A US 2007122406 A1 US2007122406 A1 US 2007122406A1
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antibody
cam
antibodies
protein
modification
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Aaron Chamberlain
John Desjarlais
Gregory Lazar
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Xencor Inc
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Xencor Inc
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Assigned to XENCOR, INC. reassignment XENCOR, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHAMBERLAIN, AARON K., DESJARLAIS, JOHN R., LAZAR, GREGORY ALAN
Publication of US20070122406A1 publication Critical patent/US20070122406A1/en
Priority to US12/724,309 priority patent/US20100311954A1/en
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    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
    • C07K16/465—Igs containing CDR-residues from one specie grafted between FR-residues from another with additional modified FR-residues
    • 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
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to optimized proteins that target the epithelial cell adhesion molecule (Ep-CAM), and their applications, particularly for therapeutic purposes.
  • Ep-CAM epithelial cell adhesion molecule
  • Epithelial cell adhesion molecule also known as epithelial glycoprotein 40 [EGP40], epithelial protein 2 [EGP-2], GA733-2, ESA, KSA, 17-1A antigen or other names
  • epithelial transmembrane protein encoded by the GA 733-2 gene
  • Ep-CAM 13:1507-1515; all expressly incorporated by reference.
  • the current model of the tertiary extracellular structure of Ep-CAM indicates the presence of three domains, including an N-terminal EGF-like domain (Armstrong, A. and Eck, S. 2003. Cancer Biol. Ther. 2: 320-325, expressly incorporated by reference), Ep-CAM is present in some normal and most neoplastic ephitelial cells (Armstrong, A. and Eck, S. 2003. Cancer Biol. Ther. 2: 320-325). Most carcinomas express Ep-CAM on their surfaces, including breast cancer, ovarian carcinoma, uterus cervix cancer, prostate cancer, kidney cancer, lung cancer, and colon cancer (Drapkin R. et al. 2004. Hum.
  • Ep-CAM Ep-CAM-like protein
  • Monoclonal antibodies are a common class of therapeutic proteins.
  • a number of favorable properties of antibodies including but not limited to specificity for target, ability to mediate immune effector mechanisms, and long half-life in serum, make antibodies powerful therapeutics.
  • a number of antibodies that target Ep-CAM have been evaluated in pre-clinical studies with cell lines and/or xenograft models or in clinical trials for the treatment of cancers.
  • These anti-EpCAM antibodies include but are not limited to MT201 (HD69 or adecatumumab; Naundorf, S. 2002. Int. J. Cancer. 100; 101-110; Prang, N. et al. 2005. Br. J. Cancer. 92: 342-349; Kunststoff, T. et al. 2001. Cancer Immunol.
  • Antibodies are immunological proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. Each chain is made up of individual immunoglobulin (Ig) domains, and thus the generic term immunoglobulin is used for such proteins. Each chain is made up of two distinct regions, referred to as the variable and constant regions. The light and heavy chain variable regions show significant sequence diversity between antibodies, and are responsible for binding the target antigen. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events.
  • IgA which includes subclasses IgA1 and IgA2
  • IgD which includes subclasses IgA1 and IgA2
  • IgE which includes subclasses IgG1, IgG2, IgG3, and IgG4
  • IgM immunoglobulin M
  • the distinguishing features between these antibody classes are their constant regions, although subtler differences may exist in the V region.
  • IgG antibodies are tetrameric proteins composed of two heavy chains and two light chains.
  • the IgG heavy chain is composed of four immunoglobulin domains linked from N- to C-terminus in the order V H -CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as V H -C ⁇ 1-C ⁇ 2-C ⁇ 3, referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain respectively).
  • the IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order V L -C L , referring to the light chain variable domain and the light chain constant domain respectively.
  • variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen.
  • the variable region is so named because it is the most distinct in sequence from other antibodies within the same class.
  • the majority of sequence variability occurs in the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the variable region outside of the CDRs is referred to as the framework (FR) region.
  • FR framework
  • this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.
  • the CDRs substantial antigen binding diversity
  • a number of high-resolution structures are available for a variety of variable region fragments from different organisms, some unbound and some in complex with antigen.
  • Fragments comprising the variable region can exist in the absence of other regions of the antibody, including for example the antigen binding fragment (Fab) comprising V H -C ⁇ 1 and V H -C L , the variable fragment (Fv) comprising V H and V L , the single chain variable fragment (scFv) comprising V H and V L linked together in the same chain, as well as a variety of other variable region fragments (Little et al., 2000, Immunol Today 21:364-370, expressly incorporated by reference).
  • Fab antigen binding fragment
  • Fv variable fragment
  • scFv single chain variable fragment
  • the Fc region of an antibody interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector functions.
  • the Fc region comprises Ig domains C ⁇ 2 and C ⁇ 3 and the N-terminal hinge leading into C ⁇ 2.
  • An important family of Fc receptors for the IgG class are the Fc gamma receptors (Fc ⁇ Rs). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290; both expressly incorporated by reference).
  • this protein family includes Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (CD32), including isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2), and Fc ⁇ RIIc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, expressly incorporated by reference).
  • These receptors typically have an extracellular domain that mediates binding to Fc, a membrane spanning region, and an intracellular domain that may mediate some signaling event within the cell.
  • These receptors are expressed in a variety of immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells.
  • NK natural killer
  • the ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells.
  • the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Ceal Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290; all expressly incorporated by reference).
  • the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is referred to as antibody dependent cell-mediated phagocytosis (ADCP).
  • ADCP antibody dependent cell-mediated phagocytosis
  • the different IgG subclasses have different affinities for the Fc ⁇ Rs, with IgG1 and IgG3 typically binding substantially better to the receptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65, expressly incorporated by reference). All Fc ⁇ Rs bind the same region on IgG Fc, yet with different affinities: the high affinity binder Fc ⁇ RI has a Kd for IgG1 of 10 ⁇ 8 M ⁇ 1 , whereas the low affinity receptors Fc ⁇ RII and Fc ⁇ RIII generally bind at 10 ⁇ 6 and 10 ⁇ 5 respectively.
  • Fc ⁇ RIIIa and Fc ⁇ RIIIb are 96% identical, however Fc ⁇ RIIIb does not have an intracellular signaling domain.
  • Fc ⁇ RI, Fc ⁇ RIIa/c, and Fc ⁇ RIIIa are positive regulators of immune complex-triggered activation, characterized by having an intracellular domain that has an immunoreceptor tyrosine-based activation motif (ITAM)
  • Fc ⁇ RIIb has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory.
  • IITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the receptors also differ in expression pattern and levels on different immune cells.
  • V158 allotype respond favorably to rituximab treatment; however, patients with the lower affinity F158 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758, expressly incorporated by reference).
  • Approximately 10-20% of humans are V158/V158 homozygous, 45% are V158/F158 heterozygous, and 35-45% of humans are F158/F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758; both expressly incorporated by reference).
  • 80-90% of humans are poor responders, that is they have at least one allele of the F158 Fc ⁇ RIIIa.
  • Fc/Fc ⁇ R binding mediates ADCC
  • Fc/C1q binding mediates complement dependent cytotoxicity (CDC).
  • a site on Fc between the C ⁇ 2 and C ⁇ 3 domains mediates interaction with the neonatal receptor FcRn, the binding of which recycles endocytosed antibody from the endosome back to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; both expressly incorporated by reference).
  • Fc to FcRn also plays a key role in antibody transport.
  • the binding site for FcRn on Fc is also the site at which the bacterial proteins A and G bind.
  • the tight binding by these proteins is typically exploited as a means to purify antibodies by employing protein A or protein G affinity chromatography during protein purification.
  • a key feature of the Fc region is the conserved N-linked glycosylation that occurs at N297. This carbohydrate, or oligosaccharide as it is sometimes referred, plays a critical structural and functional role for the antibody, and is one of the principle reasons that antibodies must be produced using mammalian expression systems.
  • an antibody-like protein that is finding an expanding role in research and therapy is the Fc fusion (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; both expressly incorporated by reference).
  • An Fc fusion is a protein wherein one or more polypeptides is operably linked to Fc.
  • An Fc fusion combines the Fc region of an antibody, and thus its favorable effector functions and pharmacokinetics, with the target-binding region of a receptor, ligand, or some other protein or protein domain. The role of the latter is to mediate target recognition, and thus it is functionally analogous to the antibody variable region. Because of the structural and functional overlap of Fc fusions with antibodies, the discussion on antibodies in the present invention extends directly to Fc fusions.
  • trastuzumab (Herceptin®, a registered trademark of Genentech), an anti-HER2/neu antibody for treatment of metastatic breast cancer, has less efficacy.
  • a promising means for enhancing the anti-tumor potency of antibodies is via enhancement of their ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC.
  • cytotoxic effector functions such as ADCC, ADCP, and CDC.
  • the importance of Fc ⁇ R-mediated effector functions for the anti-cancer activity of antibodies has been demonstrated in mice (Clynes et al., 1998, Proc Natl Acad Sci U S A 95:652-656; Clynes et al., 2000, Nat Med 6:443-446; both expressly incorporated by reference), and the affinity of interaction between Fc and certain Fc ⁇ Rs correlates with targeted cytotoxicity in cell-based assays (Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et al., 2002, Biochem Soc Trans 30:487-490; Shields et al., 2002, J Biol Chem 277:26733-26740; all
  • the balance between activating and inhibiting receptors is an important consideration, and optimal effector function may result from an antibody that has enhanced affinity for actvation receptors, for example Fc ⁇ RI, Fc ⁇ RIIa/c, and Fc ⁇ RIIIa, yet reduced affinity for the inhibitory receptor Fc ⁇ RIIb.
  • Fc ⁇ Rs can mediate antigen uptake and processing by antigen presenting cells, enhanced Fc ⁇ R affinity may also improve the capacity of antibody therapeutics to elicit an adaptive immune response.
  • ADCC has been implicated as an important effector mechanism for the anti-tumor cytotoxic capacity of some anti-Ep-CAM antibodies (Bleeker et al., 2004, J Immunol. 173(7):4699-707; Bier et al., 1998, Cancer Immunol Immunother 46:167-173, both expressly incorporated by reference).
  • the present invention provides variants of Ep-CAM targeting proteins that comprise one or more amino acid modifications that provide enhanced effector function and humanized light and heavy variable regions.
  • the present invention is directed to humanized Ep-CAM-targeting antibodies including first and/or second amino acid sequences corresponding to the heavy and light chains of the antibodies, respectively, as well as methods of using the same.
  • the first and second amino acid sequences can include sequences corresponding to CDR3, CDR2, or CDR1 of the humanized Ep-CAM antibody heavy and light chains. Such sequences can be independent, or can be combined.
  • the first and second amino acid sequences comprise a sequence corresponding to CDR3 of the humanized Ep-CAM heavy and light chains.
  • the present invention is directed to a humanized anti-Ep-CAM antibody, wherein said antibody comprises A) a first amino acid sequence comprising i) DGPWX 1 AY (SEQ ID NO:160), wherein X 1 is selected from the group consisting of F and Y; or ii) a sequence selected from the group consisting of SEQ ID NOS:129-130; and/or B) a second amino acid sequence comprising i) X 1 YSYPYT (SEQ ID NO:161), wherein X 1 is selected from the group consisting of G and Y; or ii) a sequence selected from the group consisting of SEQ ID NOS:135-136.
  • these sequences correspond to CDR3 of the heavy and light chains of the antibody.
  • the first amino acid sequence further comprises an amino acid sequence of i) X 1 X 2 FX 3 X 4 YL (SEQ ID NO:162), wherein X 1 is selected from the group consisting of Y and F; X 2 is selected from the group consisting of A and S; X 3 is selected from the group consisting of T and S; and X 4 is selected from the group consisting of N and D; and ii) a sequence selected from the group consisting of SEQ ID NOS:122-126; iii) NPGSGX 1 (SEQ ID NO:163), wherein X 1 is selected from the group consisting of G and A; iv) the sequence of SEQ ID NOS:131-132.
  • the second amino acid sequence further comprises i) X 1 NVVTY (SEQ ID NO:164), wherein X 1 is selected from the group consisting of E and Q; ii) a sequence selected from the group consisting of SEQ ID NOS: 127-128; iii) X 1 ASNRYT (SEQ ID NO:165), wherein X 1 is selected from the group consisting of G and D; or iv) an amino acid sequence selected from the group consisting of SEQ ID NOS: 133-134.
  • these sequences correspond to CDR1 and CDR2 of the heavy and light chains of the antibody.
  • first and second amino acid sequences part of the same amino acid sequence.
  • first amino acid sequence does not comprise a sequence selected from the group consisting of SEQ ID NOS: 122, 127, and 129
  • second amino acid sequence does not comprise a sequence selected from the group consisting of SEQ ID NOS: 131, 133, and 135.
  • the first amino acid sequence does not comprise SEQ ID NO:1
  • the second amino acid sequence does not comprise SEQ ID NO:105.
  • the humanized, the heavy chain variable region comprises a heavy chain framework region selected from the framework regions found in the group consisting of SEQ ID NOS:2-104.
  • the second amino acid comprises a light chain framework region selected from the framework regions found in the group consisting of SEQ ID NOS:106-121.
  • the antibody comprises a heavy chain variable region selected from the group consisting of: SEQ ID NOS: 3, 15, 27, 56 and 97, and/or the light chain variable region of SEQ ID NO:108.
  • the first amino acid sequence is selected from the group consisting of SEQ ID NOS: 2-104
  • the second amino acid sequence is selected from the group consisting of SEQ ID NOS: 106-121.
  • the antibody has an IgG1 Fc domain, or a hybrid IgG1, IgG2 Fc domain.
  • the present invention is directed to a variant anti-Ep-CAM antibody comprising a variant human Fc domain, the variant human Fc domain comprising at least one modification that alters binding of the antibody to an Fc receptor compared to a parent human Fc domain.
  • the one modification alters binding to an Fcgamma receptor.
  • the modification comprises at least one substitution selected from the group consisting of: 236A, 239D, 268E, 298A, 298D, 326D, 326E, 330L, 330Y, 332E, 333A, 334A, and 396L, wherein the numbering is according to the EU index in Kabat et al.
  • the modification includes an altered glycoform, such as defucosylation or lacking a fucose moiety.
  • the modifications can alter binding to FcRn.
  • the variant anti-Ep-CAM antibody comprise at least one modification that alters an effector function of the variant antibody compared to an unmodified anti-Ep-CAM antibody.
  • the effector function is antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytoxicity (CDC).
  • the Fc substitution comprises a substitution selected from the group consisting of: 239D and 332E, wherein the numbering is that of the EU index in Kabat et al.
  • the anti-Ep-CAM antibody comprises an Fc domain comprising at least one substitution selected from the group consisting of: K326W, K326Y, and E333S, wherein the numbering is according to the EU index in Kabat et al.
  • the modification increases the affinity of the antibody for Fc ⁇ RIIIa compared to a parent antibody. In some variations, the modification increases the affinity of the antibody for Fc ⁇ RIIIa at least 2-fold compared to a parent antibody. In other variations the modification increases the affinity of the antibody for Fc ⁇ RIIIa at least 5-fold compared to a parent antibody.
  • the Fc modification decreases the affinity of the antibody for Fc ⁇ RIIIa compared to a parent antibody. In some embodiments, the modification decreases the affinity of the antibody for Fc ⁇ RIIIa by at least 10-fold compared to a parent antibody.
  • the modification can also comprise a substitution selected from the group consisting of: 235G and 236R, wherein the numbering is that of the EU index in Kabat et al.
  • the Fc modification increases the Fc ⁇ RIIa:Fc ⁇ RIIb specificity for the antibody. In some embodiments, the modification increases the Fc ⁇ RIIa:Fc ⁇ RIIb specificity for the antibody by at least 2. In further embodiments, the modification increases the Fc ⁇ RIIa:Fc ⁇ RIIb specificity for the antibody by at least 8. In still further embodiments, the modification increases the Fc ⁇ RIIa:Fc ⁇ RIIb specificity between 7 to 11.
  • the Fc modification specifically increases maturation or activation of monocytes, macrophages, neutrophils, or dendritic cells by the antibody compared to activation of natural killer (NK) cells by the antibody.
  • the modification specifically increases activation of neutrophils by the antibody compared to activation of natural killer (NK) cells by the antibody.
  • the Fc modification not substantially increase activation of natural killer cells or specifically increases activation by the antibody of dendritic cells.
  • the modification increases binding to an activating Fc receptor and does not increase binding to Fc ⁇ RIIb.
  • the modification specifically increases monocyte or macrophage phagocytosis.
  • the present invention provides variant Ep-CAM targeting proteins that are optimized for a number of therapeutically relevant properties.
  • a variant Ep-CAM targeting protein comprises one or more amino acid modifications relative to a parent Ep-CAM targeting protein, wherein the amino acid modification(s) provide one or more optimized properties.
  • FIGS. 1A and 1B Sequences of the heavy chain variable region of the original 17-1A antibody and select antibodies of the present invention with reduced potential for immunogenicity (SEQ ID NOS:1-104 and SEQ ID NOS:122-130).
  • FIGS. 2A and 2B Sequences of the light chain variable region of the original 17-1A antibody and select antibodies of the present invention with reduced potential for immunogenicity (SEQ ID NOS:105-121 and SEQ ID NOS:13-136).
  • FIG. 3 Sequences of the constant regions of the original 17-1A antibody and select antibodies of the present invention (SEQ ID NOS:137-143).
  • FIG. 4 Expression yields of select anti-Ep-CAM antibodies of the present invention.
  • FIG. 5 SDS gels of some anti-Ep-CAM antibodies of the present invention.
  • FIG. 6 An SDS gel of an anti-Ep-CAM antibody purified after expression in lec13 cells.
  • the resulting antibody has an engineered glycoform, that is, it is defucosylated.
  • FIG. 7 Schematic representation of the AlphaScreenTM methods used to measure relative binding affinity in the present study.
  • FIG. 8 AlphaScreenTM data showing the relative binding affinity of antibodies of the present invention to the antigen, Ep-CAM, and to protein A.
  • FIG. 9 Summary of AlphaScreenTM data showing the relative binding properties of antibodies of the present invention to the antigen, Ep-CAM, and to protein A.
  • FIG. 10 AlphaScreenTM data showing the relative binding affinity of antibodies of the present invention to (7A) the antigen, Ep-CAM or (7B) the Fc gamma receptor IIIa (FcgRIIIaV).
  • FIG. 13 Binding measurements of anti-Ep-CAM proteins to Ep-CAM and to FcgammaRIIIa.
  • FIG. 15 Binding data for anti-Ep-CAM antibodies measured by surface plasmon resonance (SPR).
  • FIG. 16 Binding data for anti-Ep-CAM antibodies measured by surface plasmon resonance (SPR).
  • FIG. 20 ADCC activities of anti-Ep-CAM antibodies with (a) the LS180 cell line and (b) the LS180 and HT29 cell line.
  • FIG. 23 ADCC activity of anti-Ep-CAM antibodies with the SkBr3 cell line. Variable regions were expressed with human IgG1.
  • FIG. 26 Binding affinity of wild-type and variant Ep-CAM-targeting antibodies to various Fc ⁇ R's, Fc gamma receptors. Data shown are collected with H3.77 and L3 variable domains. Constant regions were based on either human IgG1 or a hybrid of human IgG1 and IgG2 sequences. The binding affinity is plotted as ⁇ log(KD) in molar units. Larger numbers demonstrate tighter binding and a change of 1 unit on the ordinate demonstrates a 10-fold change in binding affinity.
  • FIG. 27 Binding affinities of modified Ep-CAM-targeting antibodies to Fc receptors. Surface plasmon resonance measurements were used to test the strength of binding, which is reported as KD values in molar units. Also shown are the fold-change in binding of each antibody relative to the WT IgG1 binding affinity and the log(1/KD) values, or ⁇ log(KD), which are also plotted in FIG. 26 . The relative binding of each variant to Fc ⁇ RIIa compared to Fc ⁇ RIIB are shown in the last column. A value of zero shows equal binding of the antibody to Fc ⁇ RIIa and Fc ⁇ RIIb, whereas a value of one shows 10fold tighter binding of the antibody to Fc ⁇ RIIa than to Fc ⁇ RIIb.
  • FIG. 28 (a) Common allotypes of human IgGs. (b) Alternative allotypic versions of anti-Ep-CAM IgG antibodies.
  • FIG. 29 Sequences of Ep-CAM-targeting antibodies, including both heavy and light chain sequences (SEQ ID NOS:144-159).
  • the present invention is directed to humanized Ep-CAM-targeting antibodies including first and/or second amino acid sequences corresponding to the heavy and light chains of the antibodies, respectively, as well as methods of using the same.
  • the first and second amino acid sequences can include sequences corresponding to CDR3, CDR2, or CDR1 of the humanized Ep-CAM antibody heavy and light chains. Such sequences can be independent, or can be combined.
  • the Ep-CAM targeting antibodies can be combined with variant Fc regions designed to alter effector function, including those of U.S. patent application Ser. No. 11/124,620 filed May 5, 2005, ser. 10/822,231 filed Mar. 26, 2004, and ser. No. 10/379,392, filed Mar. 3, 2003, each of which is incorporated herein by reference in its entirety.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • ADCP antibody dependent cell-mediated phaqocytosis as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • the preferred amino acid modification herein is a substitution.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • substitution 1332E refers to a variant polypeptide, in this case an Fc variant, in which the isoleucine at position 332 is replaced with a glutamic acid.
  • amino acid and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. “analogs”, such as peptoids (see Simon et al., 1992, Proc Natl Acad Sci USA 89(20):9367) particularly when LC peptides are to be administered to a patient.
  • affinity or “binding affinity” as used herein is meant the strength of interaction between two molecules.
  • the strength of affinity is often reported with a dissociation constant, Kd or KD, such as 1*10 ⁇ 7 M, or a log(Kd), such as ⁇ 7.0, or ⁇ log(Kd), such as 7.0.
  • Kd or KD dissociation constant
  • Kd dissociation constant
  • the binding “specificity” may be defined as the relative strength of binding of a first molecule to a second molecule compared to the strength of the first molecule to a third molecule. Specificity may be reported as a ratio or quotient of binding constants for the two binding reactions. For example, a Fc ⁇ RIIa:Fc ⁇ RIIb specificity of 10 for Antibody A, means that Antibody A binds to Fc ⁇ RIIa ten-fold more strongly than it binds to Fc ⁇ RIIb.
  • An additional way to express the same Fc ⁇ RIIa:Fc ⁇ RIIb specificity for Antibody A is that the Kd of Fc ⁇ RIIb is 10-fold higher than the Kd of Fc ⁇ RIIa.
  • effector function as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC.
  • effector cell as used herein is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions.
  • Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and ⁇ T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • library herein is meant a set of Fc variants in any form, including but not limited to a list of nucleic acid or amino acid sequences, a list of nucleic acid or amino acid substitutions at variable positions, a physical library comprising nucleic acids that encode the library sequences, or a physical library comprising the Fc variant proteins, either in purified or unpurified form.
  • Ep-CAM targeting protein as used herein is meant a protein that binds to Ep-CAM, also known as epithelial glycoprotein 40 [EGP40], epithelial protein 2 [EGP-2], GA733-2, ESA, KSA, 17-1A antigen and other names.
  • the EQCAM targeting protein of the present invention may be an antibody, Fc fusion, or any other protein that binds Ep-CAM.
  • An Ep-CAM targeting protein of the present invention may bind any epitope or region on Ep-CAM, and may be specific for fragments, splice forms, or aberrent forms of Ep-CAM.
  • Preferred proteins are antibodies, including the antibodies described herein.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C ⁇ 2 and C ⁇ 3) and the hinge between Cgamma1 (C ⁇ 1) and Cgamma2 (C ⁇ 2).
  • Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below.
  • Fc polypeptide as used herein is meant a polypeptide that comprises all or part of an Fc region.
  • Fc polypeptides include antibodies, Fc fusions, isolated Fcs, and Fc fragments.
  • Fc fusion as used herein is meant a protein wherein one or more polypeptides or small molecules is operably linked to an Fc region or a derivative thereof.
  • Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; both expressly incorporated by reference).
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein or small molecule.
  • a fusion partner which in general can be any protein or small molecule.
  • the role of the non-Fc part of an Fc fusion, i.e. the fusion partner, is often but not always to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody.
  • a variety of linkers, defined and described below, may be used to covalently link Fc to a fusion partner to generate an Fc fusion.
  • Fc gamma receptor or “Sc ⁇ R” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and are substantially encoded by the Fc ⁇ R genes. In humans this family includes but is not limited to Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (CD32), including isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2), and Fc ⁇ RIIc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, expressly incorporated by reference
  • An Fc ⁇ R may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Mouse Fc ⁇ Rs include but are not limied to Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16), and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes.
  • Fc ligand as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc-ligand complex.
  • Fc ligands include but are not limited to Fc ⁇ Rs, Fc ⁇ Rs, Fc ⁇ Rs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral Fc ⁇ R.
  • IgG immunoglobulin gamma gene
  • this class comprises IgG1, IgG2, IgG3, and IgG4.
  • mice this class comprises IgG1, IgG2a, IgG2b, IgG3.
  • hybrids of IgG proteins in which amino acids for one IgG protein substituted for amino acids of a different IgG protein (e.g. IgG1/IgG2 hybrids.
  • immunoglobulin (Ig) herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • parent polypeptide or “precursor polypeptide” (including Fc parent or precursors) as used herein is meant a polypeptide that is subsequently modified to generate a variant.
  • the parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide.
  • Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.
  • parent Fc polypeptide as used herein is meant a Fc polypeptide that is modified to generate a variant
  • parent antibody as used herein is meant an antibody that is modified to generate a variant antibody.
  • positions of the Fc molecule can be altered.
  • position as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index as in Kabat. For example, position 297 is a position in the human antibody IgG1. Corresponding positions are determined as outlined above, generally through alignment with other parent sequences.
  • residue as used herein is meant a position in a protein and its associated amino acid identity.
  • Asparagine 297 also referred to as Asn297, also referred to as N297
  • Asn297 is a residue in the human antibody IgG1.
  • target antigen as used herein is meant the molecule that is bound specifically by the variable region of a given antibody.
  • a target antigen may be a protein, carbohydrate, lipid, or other chemical compound.
  • target cell as used herein is meant a cell that expresses a target antigen
  • variable region as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the V K , V ⁇ , and/or V H genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively.
  • variant protein a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification.
  • variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it.
  • the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.
  • variant polypeptide sequence herein will preferably possess at least about 80% homology with a parent polypeptide sequence, and most preferably at least about 90% homology, more preferably at least about 95% homology.
  • variant Fc or “Fc variant” as used herein is meant an Fc sequence that differs from that of a parent Fc sequence by virtue of at least one amino acid modification.
  • An Fc variant may only encompass an Fc region, or may exist in the context of an antibody, Fc fusion, or other polypeptide that is substantially encoded by Fc.
  • Fc variant may refer to the Fc polypeptide itself compositions comprising the Fc variant polypeptide, or the amino acid sequence that encodes it. Also included are Fc variants disclosed in U.S. patent application Ser.
  • variant EP-CAM targeting protein or “Eq-CAM targeting protein variant” as used herein is meant an Ep-CAM targeting protein, as defined above, that differs in sequence from that of a parent Ep-CAM targeting protein sequence by virtue of at least one amino acid modification.
  • Variant Ep-CAM targeting protein may refer to the protein itself, compositions comprising the protein, or the amino acid sequence that encodes it.
  • the present invention provides variant antibodies.
  • Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.
  • IgM has subclasses, including, but not limited to, IgM1 and IgM2.
  • isotype as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human inmunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.
  • each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • variable region three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site.
  • Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a “CDR”), in which the variation in the amino acid sequence is most significant.
  • CDR complementarity-determining region
  • each chain defines a constant region primarily responsible for effector function.
  • Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E. A. Kabat et al.).
  • immunoglobulin domains in the heavy chain.
  • immunoglobulin (Ig) domain herein is meant a region of an immunoglobutin having a distinct tertiary structure.
  • the heavy chain domains including, the constant heavy (CH) domains and the hinge domains.
  • the IgG isotypes each have three CH regions .
  • “CH” domains in the context of IgG are as follows: “CH1” refers to positions 118-220 according to the EU index as in Kabat. “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat.
  • Fc regions are the Fc regions.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and in some cases, part of the hinge.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • IgG as illustrated in FIG.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cg2 and Cg3) and the lower hinge region between Cgamma1 (Cg1) and Cgamma2 (Cg2).
  • the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below.
  • Fc polypeptide as used herein is meant a polypeptide that comprises all or part of an Fc region.
  • Fc polypeptides include antibodies, Fc fusions, isolated Fcs, and Fc fragments.
  • the antibodies are full length.
  • full length antibody herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions, including one or more modifications as outlined herein.
  • the antibodies can be a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each, respectively.
  • antibody mimetics sometimes referred to herein as “antibody mimetics”
  • chimeric antibodies humanized antibodies
  • antibody fusions sometimes referred to as “antibody conjugates”
  • Specific antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546) which consists of a single variable, (v) isolated CDR regions, (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc.
  • scFv single chain Fv molecules
  • the scaffold components can be a mixture from different species.
  • the antibody is a mixture of a human antibody and a mouse antibody, such an antibody may be a chimeric antibody and/or a humanized antibody.
  • both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species.
  • “chimeric antibodies” traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human.
  • “Humanized antibodies” generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies.
  • a humanized antibody the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs.
  • the CDRs some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs.
  • the creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536.
  • “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213).
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654.
  • Structure-based methods may be employed for humanization and affinity maturation, for example as described in U.S. Ser. No. 11/004,590. Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al. 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.
  • the antibodies of the invention multispecific antibody, and notably a bispecitic antibody, also sometimes referred to as “diabodies”. These are antibodies that bind to two (or more) different antigens. Diabodies can be manufactured in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449), e.g., prepared chemically or from hybrid hybridomas.
  • the antibody is a fully human antibody with at least one modification as outlined herein.
  • “Fully human antibody ” or “complete human antibody” refers to a human antibody having the gene sequence of an antibody derived from a human chromosome with the modifications outlined herein.
  • Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200).
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein or small molecule. Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion.
  • Protein fusion partners may include, but are not limited to, the variable region of any antibody, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • targets may be any molecule, preferably an extracellular receptor, that is implicated in disease.
  • antibody fusions include the fusion of the constant region of the heavy chain with one or more fusion partners (again including the variable region of any antibody), while other antibody fusions are substantially or completely full length antibodies with fusion partners.
  • a role of the fusion partner is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody (and in fact can be).
  • Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion (or antibody fusion).
  • Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • Such targets may be any molecule, preferably an extracellular receptor, that is implicated in disease.
  • the conjugate partner can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner.
  • linkers are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see, 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • Suitable conjugates include, but are not limited to, labels as described below, drugs and cytotoxic agents including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins.
  • cytotoxic drugs e.g., chemotherapeutic agents
  • Suitable toxins and their corresponding fragments include diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
  • Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. Additional embodiments utilize calicheamicin, auristatins, geldanamycin, maytansine, and duocarmycins and analogs; for the latter, see U.S. 2003/0050331, hereby incorporated by reference in its entirety.
  • Covalent modifications of (e.g. attachments to) antibodies are included within the scope of this invention, and are generally, but not always, done post-translationally.
  • several types of covalent attachments to the antibody are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-aminocontaining residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid: O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
  • Carboxyl side groups are selectively modified by reaction with carbodiimides (R′—N ⁇ C ⁇ N—R′), where R and R′ are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • R′ is optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Derivatization with bifunctional agents is useful for crosslinking antibodies to a water-insoluble support matrix or surface for use in a variety of methods, in addition to methods described below.
  • Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-mateimido1,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • the IgG variants disclosed herein can be modified to include one or more engineered glycoforms.
  • engineered glycoform as used herein is meant a carbohydrate composition that is covalently attached to an IgG, wherein said carbohydrate composition differs chemically from that of a parent IgG.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
  • Engineered glycoforms may be generated by a variety of methods known in the art (Uma ⁇ a et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473); (U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.
  • Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus an IgG variant, for example an antibody or Fc fusion, can include an engineered glycoform.
  • engineered glycoform may refer to the IgG variant that comprises the different carbohydrate or oligosaccharide.
  • glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are frequently the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites).
  • the antibody amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the antibody is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N- and O-linked glycosylation.
  • the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • Removal of carbohydrate moieties present on the starting antibody may be accomplished chemically or enzymatically.
  • Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact.
  • Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal. Biochem. 118:131.
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138:350.
  • Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycoside linkages. Additionally, modification of an amino acid in the glycosylation motif may be used to prevent glycosylation.
  • Another type of covalent modification of the antibody comprises linking the antibody to various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • amino acid substitutions may be made in various positions within the antibody to facilitate the addition of polymers such as PEG. See for example, U.S. Publication No. 2005/0114037, incorporated herein by reference in its entirety.
  • the covalent modification of the antibodies of the invention comprises the addition of one or more labels. In some cases, these are considered antibody fusions.
  • labelling group means any detectable label.
  • the labelling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance.
  • spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used in performing the present invention.
  • labels fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).
  • the labelling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used in performing the present invention.
  • optical dyes including, but not limited to, chromophores, phosphors and fluorophores, with the latter being specific in many instances.
  • Fluorophores can be either “small molecule” fluores, or proteinaceous fluores.
  • fluorescent label any molecule that may be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhod
  • Suitable proteinaceous fluorescent labels also include, but are not limited to, green fluorescent protein, including a Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.
  • green fluorescent protein including a Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd
  • EYFP enhanced yellow fluorescent protein
  • luciferase Rhoplasminogen activatories, Inc.
  • ⁇ galactosidase Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607
  • Renilla WO92/15673, WO95/07463, WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos.
  • antibody may mean a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa ( ⁇ ), lambda ( ⁇ ), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), sigma ( ⁇ ), and alpha ( ⁇ ) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE, and IgA (IgA1 and IgA2) isotypes respectively.
  • Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes.
  • the Ep-CAM targeting proteins of the present invention may be an antibody, referred to herein as “anti-Ep-CAM antibodies”.
  • Anti-Ep-CAM antibodies of the present invention may comprise immunoglobulin sequences that are substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including but not limited to IgG (including human subclasses IgG1, IgG2, IgG3, or IgG4 and hybrids thereof), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG, and IgM classes of antibodies. Most preferably the antibodies of the present invention comprise sequences belonging to the human IgG class of antibodies.
  • Anti-Ep-CAM antibodies of the present invention may be nonhuman, chimeric, humanized, or fully human. As will be appreciated by one skilled in the art, these different types of antibodies reflect the degree of “humaness” or potential level of immunogenicity in a human. For a description of these concepts, see Clark et al., 2000 and references cited therein (Clark, 2000, Immunol Today 21:397-402, expressly incorporated by reference). Chimeric antibodies comprise the variable region of a nonhuman antibody, for example VH and VL domains of mouse or rat origin, operably linked to the constant region of a human antibody (see for example U.S. Pat. No. 4,816,567, expressly incorporated by reference).
  • the nonhuman variable region may be derived from any organism as described above, preferably mammals and most preferably rodents or primates.
  • the antibody of the present invention comprises monkey variable domains, for example as described in Newman et al., 1992, Biotechnology 10:1455-1460, U.S. Pat. Nos. 5,658,570, and 5,750,105; all expressly incorporated by reference.
  • the variable region is derived from a nonhuman source, but its immunogenicity has been reduced using protein engineering.
  • the antibodies of the present invention are humanized (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), expressly incorporated by reference).
  • humanized antibody as used herein is meant an antibody comprising framework regions (FRs) derived from one or more human sequences and one or more complementarity determining regions (CDR's) from a non-human (usually mouse or rat) antibody.
  • CDR grafting in which a non-human antibody (the “donor”) provides the CDR's and a human antibody (the “acceptor”) provides the frameworks (Winter U.S. Pat. No.
  • the humanized antibody also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • the immunogenicity of the antibody may be reduced using a method described in U.S. Ser. No. 60/619,483, filed Oct. 14, 2004 and U.S. Ser. No. 11/004,590, entitled “Methods of Generating Variant Proteins with Increased Host String Content and Compositions Thereof”, filed on Dec. 6, 2004; both expressly incorporated by reference.
  • the antibodies of the present invention may be fully human, that is the sequences of the antibodies are completely or substantially human.
  • the Ep-CAM targeting proteins of the present invention may be an Fc fusion, referred to herein as “anti-Ep-CAM Fc fusions”.
  • Anti-Ep-CAM Fc fusions of the present invention comprise an Fc polypeptide operably linked to one or more fusion partners.
  • the role of the fusion partner typically, but not always, is to mediate binding of the Fc fusion to a target antigen. (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; both expressly incorporated by reference).
  • one of the fusion partners must bind Ep-CAM.
  • Fusion partners may be a protein, polypeptide, or small molecule. Virtually any polypeptide or molecule that targets Ep-CAM may serve as a fusion partner. Undiscovered Ep-CAM ligands may serve as fusion partners for the Ep-CAM targeting proteins of the present invention.
  • Anti-Ep-CAM Fc fusions of the invention may comprise immunoglobulin sequences that are substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including but not limited to IgG (including human subclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG, and IgM classes of antibodies. Most preferably the anti-Ep-CAM Fc fusions of the present invention comprise sequences belonging to the human IgG class of antibodies.
  • Ep-CAM targeting proteins of the present invention may comprise Fc fragments.
  • An Fc fragment of the present invention may comprise from 1-90% of the Fc region, with 10-90% being preferred, and 30-90% being most preferred.
  • an Fc fragment of the present invention may comprise an IgG1 C ⁇ 2 domain, an IgG1 C ⁇ 2 domain and hinge region, an IgG1 C ⁇ 3 domain, and so forth.
  • an Fc fragment of the present invention additionally comprises a fusion partner, effectively making it an Fc fragment fusion.
  • Fc fragments may or may not contain extra polypeptide sequence.
  • Ep-CAM targeting proteins of the present invention may be substantially encoded by genes from any organism, preferably mammals, including but not limited to humans, rodents including but not limited to mice and rats, lagomorpha including but not limited to rabbits and hares caeiidae including but not limited to camels, llamas, and dromedaries, and non-human primates, including but not limited to Prosimians, Platyrrhini (New World monkeys), Cercopithecoidea (Old World monkeys), and Hominoidea including the Gibbons and Lesser and Great Apes.
  • the Ep-CAM targeting proteins of the present invention are substantially human.
  • the Ep-CAM targeting proteins of the present invention may be substantially encoded by immunoglobulin genes belonging to any of the antibody classes.
  • the Ep-CAM targeting proteins of the present invention comprise sequences belonging to the IgG class of antibodies, including human subclasses IgG1, IgG2, IgG3, and IgG4.
  • the Ep-CAM targeting proteins of the present invention comprise sequences belonging to the IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies.
  • the Ep-CAM targeting proteins of the present invention may comprise more than one protein chain. That is, the present invention may find use in an Ep-CAM targeting protein that is a monomer or an oligomer, including a homo- or hetero-oligomer.
  • the anti-Ep-CAM antibodies and Fc fusions of the invention are based on human IgG sequences, and thus human IgG sequences are used as the “base” sequences against which other sequences are compared, including but not limited to sequences from other organisms, for example rodent and primate sequences, as well as sequences from other immunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like. It is contemplated that, although the Ep-CAM targeting proteins of the present invention are engineered in the context of one parent Ep-CAM targeting protein, the variants may be engineered in or “transferred” to the context of another, second parent Ep-CAM targeting protein.
  • first and second Ep-CAM targeting proteins typically based on sequence or structural homology between the sequences of the two Ep-CAM targeting proteins.
  • amino acid sequence of a first Ep-CAM targeting protein outlined herein is directly compared to the sequence of a second Ep-CAM targeting protein.
  • homology alignment programs known in the art for example using conserved residues as between species
  • the residues equivalent to particular amino acids in the primary sequence of the first Ep-CAM targeting protein are defined.
  • Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues.
  • Equivalent residues may also be defined by determining structural homology between a first and second Ep-CAM targeting protein that is at the level of tertiary structure for EP-CAM targeting proteins whose structures have been determined. In this case, equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the parent or precursor (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment.
  • Ep-CAM targeting proteins discovered by the present invention may be engineered into any second parent Ep-CAM targeting protein that has significant sequence or structural homology with the Ep-CAM targeting protein.
  • the variant anti-Ep-CAM antibody may be engineered in a human IgG2 parent anti-Ep-CAM antibody, a human IgA parent anti-Ep-CAM antibody, a mouse IgG2a or IgG2b parent anti-Ep-CAM antibody, and the like.
  • the context of the parent Ep-CAM targeting protein does not affect the ability to transfer the Ep-CAM targeting proteins of the present invention to other parent Ep-CAM targeting proteins.
  • the variant anti-Ep-CAM antibodies that are engineered in a human IgG1 antibody that targets one Ep-CAM epitope may be transferred into a human IgG2 antibody that targets a different Ep-CAM epitope, into an Fc fusion that comprises a human IgG1 Fc region that targets yet a different Ep-CAM epitope, and so forth.
  • Ep-CAM targeting protein of the present invention may be virtually any antibody, Fc fusion, or other protein that binds Ep-CAM.
  • Ep-CAM targeting proteins of the invention may display selectivity for Ep-CAM versus alternative targets, for example other RTKs, or selectivity for a specific form of the Ep-CAM target versus alternative forms. Examples include full-length versus splice variants, cell-surface vs. soluble forms, selectivity for various polymorphic variants, or selectivity for specific conformational forms of a target.
  • An Ep-CAM targeting protein of the present invention may bind any epitope or region on Ep-CAM, and may be specific for fragments, mutant forms, splice forms, or aberrant forms of Ep-CAM.
  • the Ep-CAM targeting proteins of the present invention may find use in a wide range of products.
  • the Ep-CAM targeting protein of the invention is a therapeutic, a diagnostic, or a research reagent.
  • the Ep-CAM targeting protein of the present invention may be used for agricultural or industrial uses.
  • An anti-Ep-CAM antibody of the present invention may find use in an antibody composition that is monoclonal or polyclonal.
  • the Ep-CAM targeting proteins of the present invention may include agonists, antagonists, neutralizing, inhibitory, or stimulatory.
  • the Ep-CAM targeting proteins of the present invention are used to kill target cells that bear the Ep-CAM target antigen, for example cancer cells.
  • the EP-CAM targeting proteins of the present invention are used to block, antagonize, or agonize the Ep-CAM target antigen.
  • the Ep-CAM targeting proteins of the present invention are used to block, antagonize, or agonize the target antigen and kill the target cells that bear the target antigen.
  • the present invention provides variant Ep-CAM targeting proteins that are optimized for a number of therapeutically relevant properties.
  • a variant Ep-CAM targeting protein comprises one or more amino acid modifications relative to a parent Ep-CAM targeting protein, wherein the amino acid modification(s) provide one or more optimized properties.
  • the Ep-CAM targeting proteins of the present invention are variants Ep-CAM targeting proteins.
  • An Ep-CAM targeting protein of the present invention differs in amino acid sequence from its parent Ep-CAM targeting protein by virtue of at least one amino acid modification.
  • variant Ep-CAM targeting proteins of the present invention have at least one amino acid modification compared to the parent.
  • the variant Ep-CAM targeting proteins of the present invention may have more than one amino acid modification as compared to the parent, for example from about one to fifty amino acid modifications, preferably from about one to ten amino acid modifications, and most preferably from about one to about five amino acid modifications compared to the parent.
  • sequences of the variant Ep-CAM targeting proteins and those of the parent Ep-CAM targeting proteins are substantially homologous.
  • the variant Ep-CAM targeting protein sequences herein will possess about 80% homology with the parent Ep-CAM targeting protein sequence, preferably at least about 90% homology, and most preferably at least about 95% homology.
  • the Ep-CAM targeting proteins of the present invention comprise amino acid modifications that provide optimized effector function properties relative to the parent. Most preferred substitutions and optimized effector function properties are described in U.S. Ser. No. 10/672,280, PCT US03/30249, and U.S. Ser. No. 10/822,231, and U.S. Ser. No. 60/627,774, filed Nov. 12, 2004 and entitled “Optimized Fc Variants”. Properties that may be optimized include but are not limited to enhanced or reduced affinity for an Fc ⁇ R.
  • the Ep-CAM targeting proteins of the present invention are optimized to possess enhanced affinity for a human activating Fc ⁇ R, preferably Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb, most preferably Fc ⁇ RIIIa.
  • the Ep-CAM targeting proteins are optimized to possess reduced affinity for the human inhibitory receptor Fc ⁇ RIIb.
  • the Ep-CAM targeting proteins of the present invention are optimized to have reduced or ablated affinity for a human Fc ⁇ R, including but not limited to Fc ⁇ RI, Fc ⁇ RIIIa, Fc ⁇ RIIb, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb. These embodiments are anticipated to provide Ep-CAM targeting proteins with enhanced therapeutic properties in humans, for example reduced effector function and reduced toxicity. In other embodiments, Ep-CAM targeting proteins of the present invention provide enhanced affinity for one or more Fc ⁇ Rs, yet reduced affinity for one or more other Fc ⁇ Rs. For example, an Ep-CAM targeting protein of the present invention may have enhanced binding to Fc ⁇ RIIIa, yet reduced binding to Fc ⁇ RIIb.
  • an Ep-CAM targeting protein of the present invention may have enhanced binding to Fc ⁇ RIIa and Fc ⁇ RI, yet reduced binding to Fc ⁇ RIIb.
  • an Ep-CAM targeting protein of the present invention may have enhanced affinity for Fc ⁇ RIIb, yet reduced affinity to one or more activating Fc ⁇ Rs.
  • the Ep-CAM targeting proteins are anti-EpCAM antibodies that comprise an Fc variant of a human Fc polypeptide as defined in U.S. patent application Ser. No. 11/124,620, or variations thereof as derived in U.S. patent application Ser. No. 11/149,943.
  • the Fc variants exhibit altered binding to an Fc ligand as compared to human Fc polypeptide.
  • the Fc variant has the formula comprising:
  • the Fc region comprises is a variant selected from the group consisting of: S239D/I332E, S239D/G236A, S239D/G236S, S239D/V264I, S239D/H268D, S239D/H268E, S239D/S298A, S239D/K326E, S239D/A330L, S239D/A330Y, S239D/A330I, I332E/V264I, I332E/H268D, I332E/H268E, I332E/S298A, I332E/K326E, I332E/A330L, I332E/A330Y, I332E/A330I, I332E/G236A, I332E/G236A, I332D/V264I, I332D/H268D, I332D/H268E, I332D/S
  • the Fc variant is selected from the group consisting of: G236S, G236A, S239D, S239E, S239N, S239Q, S239T, K246H, T260H, K246Y, D249Y, R255Y, E258Y, V264I, S267E, H268D, H268E, E272Y, E272I, E272H, K274E, G281D, E283L, E283H, S304T, S324G, S3241, K326T, A327D, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, E333Y, K334T, and K334F, wherein numbering is according to the EU index.
  • the Fc variant further comprising a substitution selected from the group consisting of S298A, K326E, K326D, K326A, E333A, and K334A, wherein numbering is according to the EU index.
  • the Fc variant comprising at least one amino acid modification in the Fc region of said parent Fc polypeptide, wherein said variant protein selectively enhances binding to one or more Fc ligands relative to one or more other Fc ligands, and wherein said Fc variant comprises a substitution at a position selected from the group consisting of: 234, 235, 236, 267, 268, 292, 293, 295, 300, 324, 327, 328, 330, and 335, wherein numbering is according to the EU index.
  • Preferred embodiments comprise optimization of Fc binding to a human Fc ⁇ R, however in alternate embodiments the Ep-CAM targeting proteins of the present invention possess enhanced or reduced affinity for Fc ⁇ Rs from nonhuman organisms, including but not limited to rodents and non-human primates. Ep-CAM targeting proteins that are optimized for binding to a nonhuman Fc ⁇ R may find use in experimentation. For example, mouse models are available for a variety of diseases that enable testing of properties such as efficacy, toxicity, and pharmacokinetics for a given drug candidate. As is known in the art, cancer cells can be grafted or injected into mice to mimic a human cancer, a process referred to as xenografting.
  • Ep-CAM targeting proteins that comprise Ep-CAM targeting proteins that are optimized for one or more mouse Fc ⁇ Rs, may provide valuable information with regard to the efficacy of the protein, its mechanism of action, and the like.
  • the EPCAM targeting proteins of the present invention may also be optimized for enhanced functionality and/or solution properties in aglycosylated form.
  • the aglycosylated Ep-CAM targeting proteins of the present invention bind an Fc ligand with greater affinity than the aglycosylated form of the parent Ep-CAM targeting protein.
  • the Fc ligands include but are not limited to Fc ⁇ Rs, C1q, FcRn, and proteins A and G, and may be from any source including but not limited to human, mouse, rat, rabbit, or monkey, preferably human.
  • the Ep-CAM targeting proteins are optimized to be more stable and/or more soluble than the aglycosylated form of the parent Ep-CAM targeting protein.
  • Ep-CAM targeting proteins of the invention may comprise modifications that modulate interaction with Fc ligands other than Fc ⁇ Rs, including but not limited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).
  • FcRHs include but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136, expressly incorporated by reference).
  • the modifications that modulate FcRn binding may be used to increase or decrease the in vivo half-life of the EP-CAM targeting protein.
  • Decreasing the in vivo half-life is useful in decreasing the toxicity of a therapeutic Ep-CAM targeting protein or in in vivo imaging procedures. More preferably, increasing the in vivo half-life is useful to increase the potency of a therapeutic Ep-CAM targeting protein and may be done by increasing the FcRn binding of the Ep-CAM targeting protein at slightly acidic pH (typically about pH 6.0).
  • slightly acidic pH typically about pH 6.0.
  • the Fc ligand specificity of the Ep-CAM targeting protein of the present invention will determine its therapeutic utility.
  • the utility of a given Ep-CAM targeting protein for therapeutic purposes will depend on the epitope or form of the Ep-CAM target antigen and the disease or indication being treated.
  • enhanced Fc ⁇ R-mediated effector functions may be preferable. This may be particularly favorable for anti-cancer Ep-CAM targeting proteins.
  • Ep-CAM targeting proteins may be used that comprise Ep-CAM targeting proteins that provide enhanced affinity for activating Fc ⁇ Rs and/or reduced affinity for inhibitory Fc ⁇ Rs.
  • Ep-CAM targeting proteins that provide differential selectivity for different activating Fc ⁇ Rs; for example, in some cases enhanced binding to Fc ⁇ RIIa and Fc ⁇ RIIIa may be desired, but not Fc ⁇ RI, whereas in other cases, enhanced binding only to Fc ⁇ RIIa may be preferred.
  • Ep-CAM targets or cancer indications it may be advantageous to reduce or ablate one or more effector functions, for example by knocking out binding to C1q, one or more Fc ⁇ R's, FcRn, or one or more other Fc ligands.
  • Ep-CAM targeting protein Clearly an important parameter that determines the most beneficial selectivity of a given Ep-CAM targeting protein to treat a given disease is the context of the Ep-CAM targeting protein, that is, what type of Ep-CAM targeting protein is being used.
  • Fc ligand selectivity or specifity of a given Ep-CAM targeting protein will provide different properties depending on whether it composes an antibody, Fc fusion, or an Ep-CAM targeting protein with a coupled fusion or conjugate partner.
  • toxin, radionucleotide, or other conjugates may be less toxic to normal cells if the Ep-CAM targeting protein that comprises them has reduced or ablated binding to one or more Fc ligands.
  • an Ep-CAM targeting protein with enhanced affinity for activating Fc ⁇ Rs such as to bind these Fc ⁇ Rs and prevent their activation.
  • an Ep-CAM targeting protein that comprises two or more Fc regions with enhanced Fc ⁇ RIIb affinity may co-engage this receptor on the surface of immune cells, thereby inhibiting proliferation of these cells.
  • an Ep-CAM targeting protein may engage its target antigen on one cell type yet engage Fc ⁇ Rs on separate cells from the target antigen, in other cases it may be advantageous to engage Fc ⁇ Rs on the surface of the same cells as the target antigen.
  • an antibody targets an antigen on a cell that also expresses one or more Fc ⁇ Rs
  • an Ep-CAM targeting protein that enhances or reduces binding to the Fc ⁇ Rs on the surface of that cell. This may be the case, for example when the Ep-CAM targeting protein is being used as an anti-cancer agent, and co-engagement of target antigen and Fc ⁇ R on the surface of the same cell promote signalling events within the cell that result in growth inhibition, apoptosis, or other anti-proliferative effect.
  • antigen and Fc ⁇ R co-engagement on the same cell may be advantageous when the Ep-CAM targeting protein is being used to modulate the immune system in some way, wherein co-engagement of target antigen and Fc ⁇ R provides some proliferative or anti-proliferative effect.
  • Ep-CAM targeting proteins that comprise two or more Fc regions may benefit from Ep-CAM targeting proteins that modulate Fc ⁇ R selectivity or specifity to co-engage Fc ⁇ Rs on the surface of the same cell.
  • the Fc ligand specificity of the Ep-CAM targeting proteins of the present invention can be modulated to create different effector function profiles that may be suited for particular Ep-CAM epitopes, indications or patient populations.
  • Table 1 describes several preferred embodiments of receptor binding profiles that include improvements to, reductions to or no effect to the binding to various receptors, where such changes may be beneficial in certain contexts.
  • the receptor binding profiles in the table could be varied by degree of increase or decrease to the specified receptors.
  • the binding changes specified could be in the context of additional binding changes to other receptors such as C1q or FcRn, for example by combining with ablation of binding to C1q to shut off complement activation, or by combining with enhanced binding to C1q to increase complement activation.
  • Fc ⁇ Rs The presence of different polymorphic forms of Fc ⁇ Rs provides yet another parameter that impacts the therapeutic utility of the Ep-CAM targeting proteins of the present invention.
  • specificity and selectivity of a given Ep-CAM targeting protein for the different classes of Fc ⁇ Rs signficantly affects the capacity of an Ep-CAM targeting protein to target a given antigen for treatment of a given disease
  • the specificity or selectivity of an Ep-CAM targeting protein for different polymorphic forms of these receptors may in part determine which research or pre-clinical experiments may be appropriate for testing, and ultimately which patient populations may or may not respond to treatment.
  • Ep-CAM targeting proteins of the present invention may be used to guide the selection of valid research and pre-clinical experiments, clinical trial design, patient selection, dosing dependence, and/or other aspects concerning clinical trials.
  • Ep-CAM targeting proteins of the present invention may be combined with other amino acid modifications in the Fc region that provide altered or optimized interaction with one or more Fc ligands, including but not limited to Fc ⁇ Rs, C1q, FcRn, FcR homologues, and/or as yet undiscovered Fc ligands. Additional modifications may provide altered or optimized affinity and/or specificity to the Fc ligands. Additional modifications may provide altered or optimized effector functions, including but not limited to ADCC, ADCP, CDC, and/or serum half-life. Such combination may provide additive, synergistic, or novel properties in antibodies or Fc fusions.
  • the Ep-CAM targeting proteins of the present invention may be combined with known Fc variants (Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J Immunol 147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci U S A 92:11980-11984; Jefferis et al., 1995, Immunol Lett 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie
  • substitutions S298A, S298D, K326E, K326D, E333A, K334A, and P396L provide optimized Fc ⁇ R binding and/or enhanced ADCC. Furthermore, as disclosed in Idusogie et al., 2001, J. Immunology 166:2571-2572, substitutions K326W, K326Y, and E333S provide enhanced binding to the complement protein C1q and enhanced CDC. Finally, as described in Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, substitutions T250Q, T250E, M428L, and M428F provide enhanced binding to FcRn and improved pharmacokinetics.
  • the Fab and hinge regions of an antibody may impact effector functions such as antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), and complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cell-mediated cytotoxicity
  • ADCP antibody dependent cell-mediated phagocytosis
  • CDC complement dependent cytotoxicity
  • anti-Ep-CAM antibodies of the present invention may comprise one or more amino acid modifications in the VL, CL, VH, CH1, and/or hinge regions of an antibody.
  • an Ep-CAM targeting protein of one antibody isotype may be engineered such that it binds to an Fc receptor of a different isotype. This may be particularly applicable when the Fc binding sites for the respective Fc receptors do not significantly overlap.
  • the structural determinants of IgA binding to Fc ⁇ RI may be engineered into an IgG Ep-CAM targeting protein.
  • Ep-CAM targeting proteins of the present invention may comprise modifications that modulate the in vivo pharmacokinetic properties of an Ep-CAM targeting protein. These include, but are not limited to, modifications that enhance affinity for the neonatal Fc receptor FcRn (U.S. Ser. No. 10/020354; WO2001US0048432; EP 2001000997063; U.S. Pat. No. 6,277,375; U.S. Ser. No. 09/933497; WO1997US0003321; U.S. Pat. No. 6,737,056; WO2000US0000973; Shields et al. J. Biol. Chem., 276(9), 6591-6604 (2001); Zhou et al. J. Mol.
  • substitutions T250Q, T250E, M428L, and M428F provide enhanced binding to FcRn and improved pharmacokinetics.
  • substitutions T250Q, T250E, M428L, and M428F provide enhanced binding to FcRn and improved pharmacokinetics.
  • preferred modifications are those that maintain the wild-type Fc's improved binding at lower pH relative to the higher pH.
  • modifications that reduce affinity for FcRn are preferred.
  • Ep-CAM targeting proteins of the present invention may comprise one or more modifications that provide optimized properties that are not specifically related to effector function per se.
  • the modifications may be amino acid modifications, or may be modifications that are made enzymatically or chemically.
  • Such modification(s) likely provide some improvement in the Ep-CAM targeting protein, for example an enhancement in its stability, solubility, function, or clinical use.
  • the present invention contemplates a variety of improvements that made be made by coupling the Ep-CAM targeting proteins of the present invention with additional modifications.
  • the Ep-CAM targeting proteins of the present invention may comprise modifications to reduce immunogenicity in humans.
  • the immunogenicity of an Ep-CAM targeting protein of the present invention is reduced using a method described in U.S. Ser. No. 60/1619,483, filed Oct. 14, 2004 and U.S. Ser. No. 11/004,590, entitled “Methods of Generating Variant Proteins with Increased Host String Content and Compositions Thereof”, filed on Dec. 6, 2004.
  • the antibodies of the present invention are humanized (Clark, 2000, Immunol Today 21:397-402, expressly incorporated by reference).
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • an immunoglobulin constant region typically that of a human immunoglobulin
  • human Fc region typically comprise a human Fc region.
  • Humanization methods include but are not limited to methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci.
  • Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973.
  • selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem.
  • Modifications to reduce immunogenicity may include modifications that reduce binding of processed peptides derived from the parent sequence to MHC proteins.
  • amino acid modifications would be engineered such that there are no or a minimal number of immune epitopes that are predicted to bind, with high affinity, to any prevalent MHC alleles.
  • Several methods of identifying MHC-binding epitopes in protein sequences are known in the art and may be used to score epitopes in an Ep-CAM targeting protein of the present invention. See for example WO 98152976; WO 02/079232; WO 0013317; U.S. Ser. Nos.
  • Sequence-based information can be used to determine a binding score for a given peptide—MHC interaction (see for example Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: p 942-948; Sturniolo et. al., 1999, Nature Biotech. 17: 555-561, all expressly incorporated by reference). It is possible to use structure-based methods in which a given peptide is computationally placed in the peptide-binding groove of a given MHC molecule and the interaction energy is determined (for example, see WO 98/59244 and WO 02/069232, both expressly incorporated by reference). Such methods may be referred to as “threading” methods.
  • MHC-binding propensity scores are calculated for each 9-residue frame along the protein sequence using a matrix method (see Sturniolo et. al., supra; Marshall et. al., 1995, J. Immunol. 154: 5927-5933, and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358; all expressly incorporated by reference).
  • the matrix comprises binding scores for specific amino acids interacting with the peptide binding pockets in different human class II MHC molecule.
  • the scores in the matrix are obtained from experimental peptide binding studies.
  • scores for a given amino acid binding to a given pocket are extrapolated from experimentally characterized alleles to additional alleles with identical or similar residues lining that pocket.
  • Matrices that are produced by extrapolation are referred to as “virtual matrices”.
  • additional amino acid modifications may be engineered to reduce the propensity of the intact molecule to interact with B cell receptors and circulating antibodies.
  • Anti-Ep-CAM antibodies and Fc fusions of the present invention may comprise amino acid modifications in one or more regions outside the Fc region, for example the antibody Fab region or the Fc fusion partner, that provide optimal properties.
  • the variable region of an antibody of the present invention may be affinity matured, that is to say that amino acid modifications have been made in the VH and/or VL domains of the antibody to enhance binding of the antibody to its target antigen.
  • modifications may be made in the Fc fusion partner to enhance affinity of the Fc fusion for its target antigen.
  • Such types of modifications may improve the association and/or the dissociation kinetics for binding to the target antigen.
  • Other modifications include those that improve selectivity for target antigen vs. alternative targets.
  • modifications that improve selectivity for antigen expressed on target vs. non-target cells include modifications that improve selectivity for antigen expressed on target vs. non-target cells.
  • Other improvements to the target recognition properties may be provided by additional modifications.
  • Such properties may include, but are not limited to, specific kinetic properties (i.e. association and dissociation kinetics), selectivity for the particular target versus alternative targets, and selectivity for a specific form of target versus alternative forms. Examples include full-length versus splice variants, cell-surface vs. soluble forms, selectivity for various polymorphic variants, or selectivity for specific conformational forms of the Ep-CAM target.
  • Ep-CAM targeting proteins of the invention may comprise one or more modifications that provide reduced or enhanced internalization of an Ep-CAM targeting protein.
  • Ep-CAM targeting proteins of the present invention can be utilized or combined with additional modifications in order to reduce the cellular internalization of an Ep-CAM targeting protein that occurs via interaction with one or more Fc ligands. This property might be expected to enhance effector function, and potentially reduce immunogenicity of the Ep-CAM targeting proteins of the invention.
  • Ep-CAM targeting proteins of the present invention can be utilized directly or combined with additional modifications in order to enhance the cellular internalization of an Ep-CAM targeting protein that occurs via interaction with one or more Fc ligands.
  • an Ep-CAM targeting protein is used that provides enhanced binding to Fc ⁇ RI, which is expressed on dendritic cells and active early in immune response.
  • This strategy could be further enhanced by combination with additional modifications, either within the Ep-CAM targeting protein or in an attached fusion or conjugate partner, that promote recognition and presentation of Fc peptide fragments by MHC molecules.
  • These strategies are expected to enhance target antigen processing and thereby improve antigenicity of the target antigen (Bonnerot and Amigorena, 1999, Immunol Rev. 172:279-84, expressly incorporated by reference), promoting an adaptive immune response and greater target cell killing by the human immune system.
  • These strategies may be particularly advantageous when the targeted antigen is shed from the cellular surface.
  • An additional application of these concepts arises with idiotype vaccine immunotherapies, in which clone-specific antibodies produced by a patient's lymphoma cells are used to vaccinate the patient.
  • modifications are made to improve biophysical properties of the Ep-CAM targeting proteins of the present invention, including but not limited to stability, solubility, and oligomeric state.
  • Modifications can include, for example, substitutions that provide more favorable intramolecular interactions in the Ep-CAM targeting protein such as to provide greater stability, or substitution of exposed nonpolar amino acids with polar amino acids for higher solubility.
  • a number of optimization goals and methods are described in U.S. Ser. No. 10/379,392, expressly incorporated by reference, that may find use for engineering additional modifications to further optimize the Ep-CAM targeting proteins of the present invention.
  • the Ep-CAM targeting proteins of the present invention can also be combined with additional modifications that reduce oligomeric state or size, such that tumor penetration is enhanced, or in vivo clearance rates are increased as desired.
  • Ep-CAM targeting proteins of the present invention include those that enable the specific formation or homodimeric or homomultimeric molecules. Such modifications include but are not limited to engineered disulfides, as well as chemical modifications or aggregation methods, which may provide a mechanism for generating covalent homodimeric or homomultimers. For example, methods of engineering and compositions of such molecules are described in Kan et al., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002, Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caron et al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J.
  • Additional modifications to the variants of the present invention include those that enable the specific formation or heterodimeric, heteromultimeric, bifunctional, and/or multifunctional molecules. Such modifications include, but are not limited to, one or more amino acid substitutions in the CH3 domain, in which the substitutions reduce homodimer formation and increase heterodimer formation. For example, methods of engineering and compositions of such molecules are described in Atwell et al., 1997, J. Mol. Biol. 270(1):26-35, and Carter et al., 2001, J. Immunol. Methods 248:7-15; both expressly incorporated by reference. Additional modifications include modifications in the hinge and CH3 domains, in which the modifications reduce the propensity to form dimers.
  • the Ep-CAM targeting proteins of the present invention comprise modifications that remove proteolytic degradation sites. These may include, for example, protease sites that reduce production yields, as well as protease sites that degrade the administered protein in vivo. In a preferred embodiment, additional modifications are made to remove covalent degradation sites such as deamidation (i.e. deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues), oxidation, and proteolytic degradation sites.
  • deamidation i.e. deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues
  • oxidation oxidation
  • Deamidation sites that are particular useful to remove are those that have enhance propensity for deamidation, including, but not limited to asparaginyl and gituamyl residues followed by glycines (NG and QG motifs, respectively). In such cases, substitution of either residue can significantly reduce the tendency for deamidation. Common oxidation sites include methionine and cysteine residues. Other covalent modifications, that can either be introduced or removed, include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains [T. E.
  • Modifications may include those that improve expression andlor purification yields from hosts or host cells commonly used for production of biologics. These include, but are not limited to various mammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines, and plants. Additional modifications include modifications that remove or reduce the ability of heavy chains to form inter-chain disulfide linkages. Additional modifications include modifications that remove or reduce the ability of heavy chains to form intra-chain disulfide linkages.
  • Ep-CAM targeting proteins of the present invention may comprise modifications that include the use of unnatural amino acids incorporated using, for example, the technologies developed by Schultz and colleagues, including but not limited to methods described by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7; expressly incorporated by reference.
  • these modifications enable manipulation of various functional, biophysical, immunological, or manufacturing properties discussed above.
  • the Ep-CAM targeting protein may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • PEG polyethylene glycol
  • Additional amino acid modifications may be made to enable specific or non-specific chemical or posttranslational modification of the Ep-CAM targeting proteins. Such modifications, include, but are not limited to PEGylation and glycosylation.
  • Specific substitutions that can be utilized to enable PEGylation include, but are not limited to, introduction of novel cysteine residues or unnatural amino acids such that efficient and specific coupling chemistries can be used to attach a PEG or otherwise polymeric moiety. Introduction of specific glycosylation sites can be achieved by introducing novel N-X-T/S sequences into the Ep-CAM targeting proteins of the present invention.
  • the Ep-CAM targeting proteins of the present invention comprise one or more engineered glycoforms.
  • engineered glycoform as used herein is meant a carbohydrate composition that is covalently attached to an Ep-CAM targeting protein, wherein the carbohydrate composition differs chemically from that of a parent Ep-CAM targeting protein.
  • An engineered protein comprising a position that lacks an attached oligosaccharide or carbohydrate may be referred to as comprising an engineered glycoform, if its parent molecule comprises an oligosaccharide, or carbohydrate at that position.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
  • Engineered glycoforms may be generated by a variety of methods known in the art (Uma ⁇ a et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S. Pat. No. 6,602,684; U.S. Ser. Nos.
  • Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus an Ep-CAM targeting protein, for example an anti-Ep-CAM antibody or Fc fusion, may comprise an engineered glycoform.
  • engineered glycoform may refer to the Ep-CAM targeting protein that comprises the different carbohydrate or oligosaccharide.
  • Ep-CAM targeting proteins of the present invention may be fused or conjugated to one or more other molecules or polypeptides.
  • Conjugate and fusion partners may be any molecule, including small molecule chemical compounds and polypeptides.
  • Possible conjugate partners include but are not limited to cytokines, cytotoxic agents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenic agents, a tyrosine kinase inhibitors. and other therapeutically active agents.
  • conjugate partners may be thought of more as payloads, that is to say that the goal of a conjugate is targeted delivery of the conjugate partner to a targeted cell, for example a cancer cell or immune cell, by the Ep-CAM targeting protein.
  • the conjugation of a toxin to an anti-Ep-CAM antibody or Fc fusion targets the delivery of the toxin to cells expressing the Ep-CAM antigen.
  • the designation of an Ep-CAM targeting protein as a fusion or conjugate is not meant to constrain it to any particular embodiment of the present invention. Rather, these terms are used loosely to convey the broad concept that any Ep-CAM targeting protein of the present invention may be linked genetically, chemically, or otherwise, to one or more polypeptides or molecules to provide some desireable property.
  • the Ep-CAM targeting proteins of the present invention are fused or conjugated to a cytokine.
  • cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators.
  • cytokines may be fused to antibody to provide an array of desireable properties. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO
  • the EpCAM targeting proteins of the present invention are fused, conjugated, or operably linked to a toxin, including but not limited to small molecule toxins and enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • a toxin including but not limited to small molecule toxins and enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • small molecule toxins include but are not limited to calicheamicin, maytansine (U.S. Pat. No. 5,208,020, expressly incorporated by reference), trichothene, and CC1065.
  • the anti-Ep-CAM antibody or Fc fusion is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antibody molecule).
  • Maytansine may, for example, be converted to May-SS-Me which may be reduced to May-SH3 and reacted with modified antibody or Fc fusion (Chari et al., 1992, Cancer Research 52: 127-131, expressly incorporated by reference) to generate a maytansinoid-antibody or maytansinoid-Fc fusion conjugate.
  • Another conjugate of interest comprises an anti-Ep-CAM antibody or Fc fusion conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • Structural analogues of calicheamicin that may be used include but are not limited to ⁇ 1 1 , ⁇ 2 1 , ⁇ 3 , N-acetyl- ⁇ 1 1 , PSAG, and ⁇ 1 1 , (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928; U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; 5,773,001; all expressly incorporated by reference).
  • Dolastatin 10 analogs such as auristatin E (AE) and monomethylauristatin E (MMAE) may find use as conjugates for the Ep-CAM targeting proteins of the present invention (Doronina et al., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65; expressly incorporated by reference).
  • AE auristatin E
  • MMAE monomethylauristatin E
  • Useful enyzmatically active toxins include but are not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordi proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • the present invention further contemplates a conjugate between an Ep-CAM targeting protein of the present invention and a compound with nucleolytic activity, for example a ribonuclease or DNA endonuclease such as a deoxyribonuclease (Dnase).
  • a compound with nucleolytic activity for example a ribonuclease or DNA endonuclease such as a deoxyribonuclease (Dnase).
  • an Ep-CAM targeting protein of the present invention may be fused, conjugated, or operably linked to a radioisotope to form a radioconjugate.
  • a radioactive isotope are available for the production of radioconjugate antibodies and Fc fusions. Examples include, but are not limited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu. See for example, reference.
  • an targeti Ep-CAM ng protein of the present invention may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the Ep-CAM targeting protein-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide).
  • a “ligand” e.g. avidin
  • a cytotoxic agent e.g. a radionucleotide
  • the Ep-CAM targeting protein is conjugated or operably linked to an enzyme in order to employ Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT).
  • ADPT Antibody Dependent Enzyme Mediated Prodrug Therapy
  • ADEPT may be used by conjugating or operably linking the Ep-CAM targeting protein to a prodrug-activating enzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see PCT WO 81/01145) to an active anti-cancer drug.
  • a prodrug e.g. a peptidyl chemotherapeutic agent, see PCT WO 81/01145
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include but are not limited to alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as .beta.-galactosidase and neuramimidase useful for converting glycosylated prodrugs into free drugs; beta-
  • Ep-CAM targeting protein-abzyme conjugates can be prepared for delivery of the abzyme to a tumor cell population.
  • additional conjugates are contemplated for the Ep-CAM targeting proteins of the present invention.
  • chemotherapeutic agents, anti-angiogenic agents, tyrosine kinase inhibitors, and other therapeutic agents are described below, which may find use as Ep-CAM targeting protein conjugates.
  • an Ep-CAM targeting protein may be a multimeric Fc polypeptide, comprising two or more Fc regions.
  • Fc regions may be linked using a chemical engineering approach.
  • Fab's and Fc's may be linked by thioether bonds originating at cysteine residues in the hinges, generating molecules such as FabFc 2 (Kan et al., 2001, J. Immunol, 2001, 166: 1320-1326; Stevenson et a., 2002, Recent Results Cancer Res. 159: 104-12; U.S. Pat.
  • Fc regions may be linked using disulfide engineering and/or chemical cross-linking, for example as described in Caron et al., 1992, J. Exp, Med. 176:1191-1195, and Shopes, 1992, J. Immunol. 148(9):2918-22; both expressly incorporated by reference.
  • Fc regions may be linked genetically, For example multiple C ⁇ 2 domains have been fused between the Fab and Fc regions of an antibody (White et al., 2001, Protein Expression and Purification 21: 446-455, expressly incorporated by reference).
  • Fc regions in an Ep-CAM targeting protein are linked genetically to generated tandemly linked Fc regions as described in U.S. Ser. No. 60/531,752, filed Dec. 22, 2003, entitled “Fc polypeptides with novel Fc receptor binding sites”, expressly incorporated by reference.
  • Tandemly linked Fc polypeptides may comprise two or more Fc regions, preferably one to three, most preferably two Fc regions. It may be advantageous to explore a number of engineering constructs in order to obtain homo- or hetero- tandemly linked Ep-CAM targeting proteins with the most favorable structural and functional properties.
  • Tandemly linked Ep-CAM targeting proteins may be homo- tandemly linked Ep-CAM targeting proteins, that is an Ep-CAM targeting protein of one isotype is fused genetically to another Ep-CAM targeting protein of the same isotype. It is anticipated that because there are multiple Fc ⁇ R, C1q, and/or FcRn binding sites on tandemly linked Fc polypeptides, effector functions and/or pharmacokinetics may be enhanced.
  • Ep-CAM targeting proteins from different isotypes may be tandemly linked, referred to as hetero-tandemly linked Ep-CAM targeting proteins. For example, because of the capacity to target Fc ⁇ R and Fc ⁇ RI receptors, an Ep-CAM targeting protein that binds both Fc ⁇ Rs and Fc ⁇ RI may provide a significant clinical improvement.
  • Fusion and conjugate partners may be linked to any region of an Ep-CAM targeting protein of the present invention, including at the N- or C-termini, or at some residue in-between the termini.
  • a fusion or conjugate partner is linked at the N- or C-terminus of the Ep-CAM targeting protein, most preferably the Nterminus.
  • linkers may find use in the present invention to covalently link Ep-CAM targeting proteins to a fusion or conjuate partner or generate an Fc fusion.
  • linker By “linker”, “linker sequence”, “spacer”, “tethering sequence” or grammatical equivalents thereof, herein is meant a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and often serves to place the two molecules in a preferred configuration.
  • a number of strategies may be used to covatently link molecules together. These include, but are not limited to polypeptide linkages between N- and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents.
  • the linker is a peptide bond, generated by recombinant techniques or peptide synthesis.
  • linker for a specific case where two polypeptide chains are to be connected depends on various parameters, including but not limited to the nature of the two polypeptide chains (e.g., whether they naturally oligomerize), the distance between the N- and the C-termini to be connected if known, and/or the stability of the linker towards proteolysis and oxidation.
  • the linker may contain amino acid residues that provide flexibility.
  • the linker peptide may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
  • the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • Suitable lengths for this purpose include at least one and not more than 50 amino acid residues
  • the linker is from about 1 to 30 amino acids in length, with linkers of 1 to 20 amino acids in length being most preferred.
  • the amino acid residues selected for inclusion in the linker peptide should exhibit properties that do not interfere significantly with the activity of the polypeptide.
  • the linker peptide on the whole should not exhibit a charge that would be inconsistent with the activity of the polypeptide, or interfere with internal folding, or form bonds or other interactions with amino acid residues in one or more of the monomers that would seriously impede the binding of receptor monomer domains.
  • Useful linkers include glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:168), (GGGGS)n (SEQ ID NO:169), and (GGGS)n (SEQ ID NO:170), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art. Glycine-serine polymers are preferred since both of these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
  • Linkers may also be identified by screening databases of known three-dimensional structures for naturally occurring motifs that can bridge the gap between two polypeptide chains.
  • the linker is not immunogenic when administered in a human patient.
  • linkers may be chosen such that they have low immunogenicity or are thought to have low immunogenicity.
  • a linker may be chosen that exists naturally in a human.
  • the linker has the sequence of the hinge region of an antibody, that is the sequence that links the antibody Fab and Fc regions; alternatively the linker has a sequence that comprises part of the hinge region, or a sequence that is substantially similar to the hinge region of an antibody.
  • Another way of obtaining a suitable linker is by optimizing a simple linker, e.g., (Gly4Ser)n, through random mutagenesis.
  • additional linker polypeptides can be created to select amino acids that more optimally interact with the domains being linked.
  • Other types of linkers that may be used in the present invention include artificial polypeptide linkers and inteins.
  • disulfide bonds are designed to link the two molecules.
  • linkers are chemical cross-linking agents.
  • bifunctional protein coupling agents including but not limited to N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,
  • a ricin immunotoxin can be prepared as described in Vitetta et al., 1971, Science 238:1098, expressly incorporated by reference.
  • Chemical linkers may enable chelation of an isotope.
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid is an exemplary chelating agent for conjugation of radionucteotide to the antibody (see PCT WO 94/11026).
  • the linker may be cleavable, facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker for example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al., 1992, Cancer Research 52: 127-131, expressly incorporated by reference) may be used.
  • a variety of nonproteinaceous polymers including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is may find use to link the Ep-CAM targeting proteins of the present invention to a fusion or conjugate partner to generate an anti-Ep-CAM Fc fusion, or to link the Ep-CAM targeting proteins of the present invention to a conjugate.
  • the present invention provides methods for producing and experimentally testing Ep-CAM targeting proteins.
  • the described methods are not meant to constrain the present invention to any particular application or theory of operation. Rather, the provided methods are meant to illustrate generally that one or more Ep-CAM targeting proteins may be produced and experimentally tested to obtain variant Ep-CAM targeting proteins.
  • nucleic acids are created that encode the Ep-CAM targeting proteins, and that may then be cloned into host cells, expressed and assayed, if desired.
  • nucleic acids, and particularly DNA may be made that encode each protein sequence.
  • Such methods include but are not limited to gene assembly methods, PCR-based method and methods which use variations of PCR, ligase chain reaction-based methods, pooled oligo methods such as those used in synthetic shuffling, error-prone amplification methods and methods which use oligos with random mutations, classical site-directed mutagenesis methods, cassette mutagenesis, and other amplification and gene synthesis methods.
  • gene assembly methods PCR-based method and methods which use variations of PCR
  • ligase chain reaction-based methods pooled oligo methods such as those used in synthetic shuffling
  • error-prone amplification methods and methods which use oligos with random mutations
  • classical site-directed mutagenesis methods cassette mutagenesis
  • cassette mutagenesis cassette mutagenesis
  • other amplification and gene synthesis methods include but are not limited to gene assembly methods, PCR-based method and methods which use variations of PCR, ligase chain reaction-based methods, pooled oligo methods such as those used in synthetic shuff
  • the Ep-CAM targeting proteins of the present invention may be produced by culturing a host cell transformed with nucleic acid, preferably an expression vector, containing nucleic acid encoding the Ep-CAM targeting proteins, under the appropriate conditions to induce or cause expression of the protein.
  • the conditions appropriate for expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • a wide variety of appropriate host cells may be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast.
  • a variety of cell lines that may find use in the present invention are described in the ATCC® cell line catalog, available from the American Type Culture Collection.
  • the Ep-CAM targeting proteins are expressed in mammalian expression systems, including systems in which the expression constructs are introduced into the mammalian cells using virus such as retrovirus or adenovirus.
  • Any mammalian cells may be used, with human, mouse, rat, hamster, and primate cells being particularly preferred. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cells and variants thereof.
  • library proteins are expressed in bacterial cells.
  • Bacterial expression systems are well known in the art, and include Escherichia coli ( E.
  • Ep-CAM targeting proteins are produced in insect cells (e.g. sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S. cerevsiae, Picha, etc).
  • Ep-CAM targeting proteins are expressed in vitro using cell free translation systems. In vitro translation systems derived from both prokaryotic (e.g. E. coli ) and eukaryotic (e.g. wheat germ, rabbit reticulocytes) cells are available and may be chosen based on the expression levels and functional properties of the protein of interest.
  • Ep-CAM targeting proteins may be produced by chemical synthesis methods.
  • transgenic expression systems both animal (e.g. cow, sheep or goat milk, embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.)
  • the nucleic acids that encode the Ep-CAM targeting proteins of the present invention may be incorporated into an expression vector in order to express the protein.
  • a variety of expression vectors may be utilized for protein expression.
  • Expression vectors may comprise self-replicating extra-chromosomal vectors or vectors which integrate into a host genome. Expression vectors are constructed to be compatible with the host cell type.
  • expression vectors which find use in the present invention include but are not limited to those which enable protein expression in mammalian cells, bacteria insect cells, yeast, and in in vitro systems.
  • a variety of expression vectors are available, commercially or otherwise, that may find use in the present invention for expressing Ep-CAM targeting proteins.
  • Expression vectors typically comprise a protein operably linked with control or regulatory sequences, selectable markers, any fusion partners, and/or additional elements.
  • operably linked herein is meant that the nucleic acid is placed into a functional relationship with another nucleic acid sequence.
  • these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the Ep-CAM targeting protein, and are typically appropriate to the host cell used to express the protein.
  • the transcriptional and translational regulatory sequences may include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • expression vectors typically contain a selection gene or marker to allow the selection of transformed host cells containing the expression vector. Selection genes are well known in the art and will vary with the host cell used.
  • Ep-CAM targeting proteins may be operably linked to a fusion partner to enable targeting of the expressed protein, purification, screening, display, and the like. Fusion partners may be linked to the Ep-CAM targeting protein sequence via a linker sequences.
  • the linker sequence will generally comprise a small number of amino acids, typically less than ten, although longer linkers may also be used. Typically, linker sequences are selected to be flexible and resistant to degradation. As will be appreciated by those skilled in the art, any of a wide variety of sequences may be used as linkers.
  • a common linker sequence comprises the amino acid sequence GGGGS (SEQ ID NO:169).
  • a fusion partner may be a targeting or signal sequence that directs Ep-CAM targeting protein and any associated fusion partners to a desired cellular location or to the extracellular media.
  • certain signaling sequences may target a protein to be either secreted into the growth media, or into the periplasmic space, located between the inner and outer membrane of the cell.
  • a fusion partner may also be a sequence that encodes a peptide or protein that enables purification and/or screening.
  • Such fusion partners include but are not limited to polyhistidine tags (His-tags) (for example H 6 and H 10 or other tags for use with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g.
  • an Ep-CAM targeting protein may be purified using a His-tag by immobilizing it to a Ni +2 affinity column, and then after purification the same His-tag may be used to immobilize the antibody to a Ni +2 coated plate to perform an ELISA or other binding assay (as described below).
  • a fusion partner may enable the use of a selection method to screen Ep-CAM targeting proteins (see below). Fusion partners that enable a variety of selection methods are well-known in the art, and all of these find use in the present invention. For example, by fusing the members of an Ep-CAM targeting protein library to the gene III protein, phage display can be employed (Kay et at. Phage display of peptides and proteins: a laboratory manual, Academic Press, San Diego, Calif., 1996; Lowman et al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science 228:1315-1317; all expressly incorporated by reference). Fusion partners may enable Ep-CAM targeting proteins to be labeled.
  • a fusion partner may bind to a specific sequence on the expression vector, enabling the fusion partner and associated Ep-CAM targeting protein to be linked covalently or noncovalently with the nucleic acid that encodes them.
  • a fusion partner may bind to a specific sequence on the expression vector, enabling the fusion partner and associated Ep-CAM targeting protein to be linked covalently or noncovalently with the nucleic acid that encodes them.
  • transfection may be either transient or stable.
  • Ep-CAM targeting proteins are purified or isolated after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins can find use in the present invention for purification of Ep-CAM targeting proteins.
  • the bacterial proteins A and G bind to the Fc region.
  • the bacterial protein L binds to the Fab region of some antibodies, as of course does the antibody's target antigen.
  • Purification can often be enabled by a particular fusion partner.
  • Ep-CAM targeting proteins may be purified using glutathione resin if a GST fusion is employed, Ni +2 affinity chromatography if a His-tag is employed, or immobilized anti-flag antibody if a flag-tag is used.
  • suitable purification techniques see Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994. The degree of purification necessary will vary depending on the screen or use of the Ep-CAM targeting proteins.
  • no purification is necessary.
  • screening may take place directly from the media.
  • some methods of selection do not involve purification of proteins.
  • protein purification may not be performed.
  • Ep-CAM targeting proteins may be screened using a variety of methods, including but not limited to those that use in vitro assays, in vivo and cell-based assays, and selection technologies. Automation and high-throughput screening technologies may be utilized in the screening procedures. Screening may employ the use of a fusion partner or label. The use of fusion partners has been discussed above.
  • label herein is meant that the Ep-CAM targeting proteins of the invention have one or more elements, isotopes, or chemical compounds attached to enable the detection in a screen.
  • labels fall into three classes: a) immune labels, which may be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, and c) small molecule labels, which may include fluorescent and colorimetric dyes, or molecules such as biotin that enable other labeling methods. Labels may be incorporated into the compound at any position and may be incorporated in vitro or in vivo during protein expression.
  • the functional and/or biophysical properties of Ep-CAM targeting proteins are screened in an in vitro assay.
  • In vitro assays may allow a broad dynamic range for screening properties of interest.
  • Properties of Ep-CAM targeting proteins that may be screened include but are not limited to stability, solubility, and affinity for Fc ligands, for example Fc ⁇ Rs. Multiple properties may be screened simultaneously or individually. Proteins may be purified or unpurified, depending on the requirements of the assay.
  • the screen is a qualitative or quantitative binding assay for binding of Ep-CAM targeting proteins to a protein or nonprotein molecule that is known or thought to bind the Ep-CAM targeting protein.
  • the screen is a binding assay for measuring binding to the Ep-CAM target antigen.
  • the screen is an assay for binding of Ep-CAM targeting proteins to an Fc ligand, including but are not limited to the family of Fc ⁇ Rs, the neonatal receptor FcRn, the complement protein C1q, and the bacterial proteins A and G.
  • the Fc ligands may be from any organism, with humans, mice, rats, rabbits, and monkeys preferred.
  • Binding assays can be carried out using a variety of methods known in the art, including but not limited to FRET (Fluorescence Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreenTM (Amplified Luminescent Proximity Homogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface Plasmon Resonance, also known as BIACORE®), isothermal titration calorimetry, differential scanning calorimetry, gel electrophoresis, and chromatography including gel filtration. These and other methods may take advantage of some fusion partner or label of the Ep-CAM targeting protein. Assays may employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • Ep-CAM targeting proteins for example stability and solubility
  • Protein stability may be determined by measuring the thermodynamic equilibrium between folded and unfolded states.
  • Ep-CAM targeting proteins of the present invention may be unfolded using chemical denaturant, heat, or pH, and this transition may be monitored using methods including but not limited to circular dichroism spectroscopy, fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy, calorimetry, and proteolysis.
  • the kinetic parameters of the folding and unfolding transitions may also be monitored using these and other techniques.
  • Ep-CAM targeting protein may be quantitatively or qualitatively determined using a wide range of methods that are known in the art, Methods which may find use in the present invention for characterizing the biophysical properties of Ep-CAM targeting proteins include gel electrophoresis, isoelectric focusing, capillary electrophoresis, chromatography such as size exclusion chromatography, ion-exchange chromatography, and reversed-phase high performance liquid chromatography, peptide mapping, oligosaccharide mapping, mass spectrometry, ultraviolet absorbance spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, analytical ultra-centrifugation, dynamic light scattering, proteolysis, and cross-linking, turbidity measurement, filter retardation assays, immunological assays, fluorescent dye binding assays, protein-staining assays microscopy, and detection of aggregates via ELISA or other binding
  • Structural analysis employing X-ray crystallographic techniques and NMR spectroscopy may also find use.
  • stability and/or solubility may be measured by determining the amount of protein solution after some defined period of time.
  • the protein may or may not be exposed to some extreme condition, for example elevated temperature, low pH, or the presence of denaturant.
  • the aforementioned functional and binding assays also provide ways to perform such a measurement.
  • a solution comprising an Ep-CAM targeting protein could be assayed for its ability to bind target antigen, then exposed to elevated temperature for one or more defined periods of time, then assayed for antigen binding again. Because unfolded and aggregated protein is not expected to be capable of binding antigen, the amount of activity remaining provides a measure of the Ep-CAM targeting protein's stability and solubility.
  • the library is screened using one or more cell-based or in vitro assays.
  • Ep-CAM targeting proteins purified or unpurified, are typically added exogenously such that cells are exposed to individual variants or groups of variants belonging to a library.
  • These assays are typically, but not always, based on the biology of the ability of the anti-Ep-CAM antibody or Fc fusion to bind to Ep-CAM and mediate some biochemical event, for example effector functions like cellular lysis, phagocytosis, ligand/receptor binding inhibition, inhibition of growth and/or proliferation, and the like.
  • Such assays often involve monitoring the response of cells to Ep-CAM targeting protein, for example cell survival, cell death, cellular phagocytosis, cell lysis, change in cellular morphology, or transcriptional activation such as cellular expression of a natural gene or reporter gene.
  • such assays may measure the ability of Ep-CAM targeting proteins to elicit ADCC, ADCP, or CDC.
  • additional cells or components that is in addition to the target cells, may need to be added, for example example serum complement, or effector cells such as peripheral blood monocytes (PBMCs), NK cells, macrophages, and the like.
  • PBMCs peripheral blood monocytes
  • NK cells macrophages, and the like.
  • additional cells may be from any organism, preferably humans, mice, rat, rabbit, and monkey.
  • Crosslinked or monomeric antibodies and Fc fusions may cause apoptosis of certain cell lines expressing the antibody's target antigen, or they may mediate attack on target cells by immune cells which have been added to the assay.
  • Methods for monitoring cell death or viability include the use of dyes, fluorophores, immunochemical, cytochemical, and radioactive reagents.
  • caspase assays or annexin-flourconjugates may enable apoptosis to be measured, and uptake or release of radioactive substrates (e.g. Chromium-51 release assays) or the metabolic reduction of fluorescent dyes such as alamar blue may enable cell growth, proliferationor activation to be monitored.
  • the DELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, MA.) is used.
  • dead or damaged target cells may be monitoried by measuring the release of one or more natural intracellular proteins, for example lactate dehydrogenase.
  • Transcriptional activation may also serve as a method for assaying function in cell-based assays.
  • response may be monitored by assaying for natural genes or proteins which may be upregulated or down-regulated, for example the release of certain interleukins may be measured, or alternatively readout may be via a luciferase or GFP-reporter construct.
  • Cell-based assays may also involve the measure of morphological changes of cells as a response to the presence of an EP-CAM targeting protein.
  • Cell types for such assays may be prokaryotic or eukaryotic, and a variety of cell lines that are known in the art may be employed.
  • cell-based screens are performed using cells that have been transformed or transfected with nucleic acids encoding the Ep-CAM targeting proteins.
  • In vitro assays include but are not limited to binding assays, ADCC, CDC, cytotoxicity, proliferation, peroxide/ozone release, chemotaxis of effector cells, inhibition of such assays by reduced effector function antibodies; ranges of activities such as >100 ⁇ improvement or >100 ⁇ reduction, blends of receptor activation and the assay outcomes that are expected from such receptor profiles.
  • Ep-CAM targeting proteins of the present invention may be characterized in cell, tissue, and whole organism experiments.
  • drugs are often tested in animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure a drug's efficacy for treatment against a disease or disease model, or to measure a drug's pharmacokinetics, toxicity, and other properties.
  • the animals may be referred to as disease models.
  • Ep-CAM targeting proteins of the present invention a particular challenge arises when using animal models to evaluate the potential for in-human efficacy of candidate polypeptides—this is due, at least in part, to the fact that Ep-CAM targeting proteins that have a specific effect on the affinity for a human Fc receptor may not have a similar affinity effect with the orthologous animal receptor.
  • Ep-CAM targeting proteins that have a specific effect on the affinity for a human Fc receptor may not have a similar affinity effect with the orthologous animal receptor.
  • These problems can be further exacerbated by the inevitable ambiguities associated with correct assignment of true orthologues (Mechetina et al., Immunogenetics, 2002 54:463-468, expressly incorporated by reference), and the fact that some orthologues simply do not exist in the animal (e g. humans possess an FcRIIa whereas mice do not).
  • Therapeutics are often tested in mice, including but not limited to nude mice, SCID mice, xenograft mice, and transgenic mice (including knockins and knockouts).
  • an anti-Ep-CAM antibody or Fc fusion of the present invention that is intended as an anti-cancer therapeutic may be tested in a mouse cancer model, for example a xenograft mouse.
  • a tumor or tumor cell line is grafted onto or injected into a mouse, and subsequently the mouse is treated with the therapeutic to determine the ability of the anti-Ep-CAM antibody or Fc fusion to reduce or inhibit cancer growth and metastasis.
  • An alternative approach is the use of a SCID murine model in which immune-deficient mice are injected with human PBLs, conferring a semi-functional and human immune—system with an appropriate array of human FcRs—to the mice that have subsequently been injected with antibodies or Fc-polypeptides that target injected human tumor cells.
  • the Fc-polypeptides that target the desired antigen such as her2/neu on SkOV3 ovarian cancer cells
  • Such experimentation may provide meaningful data for determination of the potential of the Ep-CAM targeting protein to be used as a therapeutic, Any organism, preferably mammals, may be used for testing.
  • monkeys can be suitable therapeutic models, and thus may be used to test the efficacy, toxicity, pharmacokinetics, or other property of the anti-Ep-CAM antibodies and Fc fusions of the present invention.
  • Tests of the Ep-CAM targeting proteins of the present invention in humans are ultimately required for approval as drugs, and thus of course these experiments are contemplated.
  • the Ep-CAM targeting proteins of the present invention may be tested in humans to determine their therapeutic efficacy, toxicity, pharmacokinetics, and/or other clinical properties.
  • Ep-CAM targeting proteins of the present invention may confer superior performance on Fc-containing therapeutics in animal models or in humans.
  • the receptor binding profiles of such Ep-CAM targeting proteins, as described in this specification, may, for example, be selected to increase the potency of cytotoxic drugs or to target specific effector functions or effector cells to improve the selectivity of the drug's action.
  • receptor binding profiles can be selected that may reduce some or all effector functions thereby reducing the side-effects or toxicity of such Fc-containing drug.
  • an Ep-CAM targeting protein with reduced binding to Fc ⁇ RIIIa, Fc ⁇ RI and Fc ⁇ RIIa can be selected to eliminate most cell-mediated effector function, or an Ep-CAM targeting protein with reduced binding to C1q may be selected to limit complement-mediated effector functions.
  • such effector functions are known to have potential toxic effects, therefore eliminating them may increase the safety of the Fc-bearing drug and such improved safety may be characterized in animal models.
  • such effector functions are known to mediate the desirable therapeutic activity, therefore enhancing them may increase the activity or potency of the Fc-bearing drug and such improved activity or potency may be characterized in animal models.
  • Optimized Ep-CAM targeting proteins can be tested in a variety of orthotopic tumor models. These clinically relevant animal models are important in the study of pathophysiology and therapy of aggressive cancers like pancreatic, prostate and breast cancer. Immune deprived mice including, but not limited to athymic nude or SCID mice are frequently used in scoring of local and systemic tumor spread from the site of intraorgan (e.g. pancreas, prostate or mammary gland) injection of human tumor cells or fragments of donor patients.
  • intraorgan e.g. pancreas, prostate or mammary gland
  • EP-CAM targeting proteins of the present invention may be assessed for efficacy in clinically relevant animal models of various human diseases.
  • relevant models include various transgenic animals for specific tumor antigens
  • Ep-CAM targeting proteins of the present invention may confer superior activity on Fc-containing drugs in such transgenic models, in particular variants with binding profiles optimized for human Fc ⁇ RIIIa mediated activity may show superior activity in transgenic CD16 mice. Similar improvements in efficacy in mice transgenic for the other human Fc receptors, e.g. Fc ⁇ RIIa, Fc ⁇ RI, etc., may be observed for Ep-CAM targeting proteins with binding profiles optimized for the respective receptors. Mice transgenic for multiple human receptors would show improved activity for Ep-CAM targeting proteins with binding profiles optimized for the corresponding multiple receptors, for example as outlined in Table 1.
  • proxy molecules may find utility as proxies for assessing potential in-human efficacy.
  • Such proxy molecules would preferably mimic—in the animal system—the FcR and/or complement biology of a corresponding candidate human Ep-CAM targeting protein. This mimicry is most likely to be manifested by relative association affinities between specific Ep-CAM targeting proteins and animal vs. human receptors. For example, if one were using a mouse model to assess the potential in-human efficacy of an Ep-CAM targeting protein that has enhanced affinity for human FcRIIIa, an appropriate proxy variant would have enhanced affinity for mouse FcRIII-2 (mouse CD16-2).
  • an appropriate proxy variant would have reduced affinity for mouse FcRII.
  • the proxy Ep-CAM targeting proteins could be created in the context of a human Ep-CAM targeting protein, an animal Ep-CAM targeting protein, or both.
  • the testing of Ep-CAM targeting proteins may include study of efficacy in primates (e.g. cynomolgus monkey model) to facilitate the evaluation of depletion of specific target cells harboring Ep-CAM antigen.
  • primates e.g. cynomolgus monkey model
  • Additional primate models include but not limited to that of the rhesus monkey and Fc polypetides in therapeutic studies of autoimmune, transplantation and cancer,
  • Toxicity studies are performed to determine the antibody or Fc-fusion related-effects that cannot be evaluated in standard pharmacology profile or occur only after repeated administration of the agent. Most toxicity tests are performed in two species—a rodent and a non-rodent—to ensure that any unexpected adverse effects are not overlooked before new therapeutic entities are introduced into man. In general, these models may measure a variety of toxicities including genotoxicity, chronic toxicity, immunogenicity, reproductive/developmental toxicity and carcinogenicity. Included within the aforementioned parameters are standard measurement of food consumption, bodyweight, antibody formation, clinical chemistry, and macro- and microscopic examination of standard organs/tissues (e.g. cardiotoxicity). Additional parameters of measurement are injection site trauma and the measurement of neutralizing antibodies, if any.
  • PK pharmacokinetics
  • the pharmacokinetics (PK) of the Ep-CAM targeting proteins of the invention can be studied in a variety of animal systems, with the most relevant being non-human primates such as the cynomolgus, rhesus monkeys.
  • Single or repeated i.v. or s.c. administrations over a dose range of 6000-fold (0.05-300 mg/kg) can be evaluated for the half-life (days to weeks) using plasma concentration and clearance as well as volume of distribution at a steady state and level of systemic absorbance can be measured.
  • Examples of such parameters of measurement generally include maximum observed plasma concentration (Cmax), the time to reach Cmax (Tmax), the area under the plasma concentration-time curve from time 0 to infinity [AUC(0-inf] and apparent elimination half-life (T1/2). Additional measured prameters could include compartmental analysis of concentration-time data obtained following i.v. administration and bioavailability.
  • Examples of pharmacological/toxicological studies using cynomolgus have been established for Rituxan and Zevatin in which monoclonal antibodies to CD20 are cross-reactive. Biodistribution, dosimetry (for radiolabled antibodies or Fc fusions), and PK studies can also be done in rodent models. Such studies would evaluate tolerance at all doses administered, toxicity to local tissues, preferential localization to rodent xenograft animal models, depletion of target cells (e.g. CD20 positive cells).
  • target cells e.g. CD20 positive cells
  • Ep-CAM targeting proteins of the present invention may confer superior pharmacokinetics on Fc-containing therapeutics in animal systems or in humans.
  • increased binding to FcRn may increase the half-life and exposure of the Fc-containing drug.
  • decreased binding to FcRn may decrease the half-life and exposure of the Fc-containing drug in cases where reduced exposure is favorable such as when such drug has side-effects.
  • Fc receptors are differentially expressed on various immune cell types, as well as in different tissues. Differential tissue distribution of Fc receptors may ultimately have an impact on the pharmacodynamic (PD) and pharmacokinetic (PK) properties of EP-CAM targeting proteins of the present invention. Because Ep-CAM targeting proteins of the presentation have varying affinities for the array of Fc receptors, further screening of the polypeptides for PD and/or PK properties may be extremely useful for defining the optimal balance of PD, PK, and therapeutic efficacy conferred by each candidate polypeptide.
  • PD pharmacodynamic
  • PK pharmacokinetic
  • Pharmacodynamic studies may include, but are not limited to, targeting specific tumor cells or blocking signaling mechanisms, measuring depletion of target antigen expressing cells or signals, etc.
  • the Ep-CAM targeting proteins of the present invention may target particular effector cell populations and thereby direct Fc-containing drugs to recruit certain activities to improve potency or to increase penetration into a particularly favorable physiological compartment.
  • neutrophil activity and localization can be targeted by an Ep-CAM targeting protein that preferentially targets Fc ⁇ RIIIb.
  • Such pharmacodynamic effects may be demonstrated in animal models or in humans.
  • Ep-CAM targeting proteins of the present invention may be used for various therapeutic purposes. As will be appreciated by those skilled in the art, the Ep-CAM targeting proteins of the present invention may be used for any therapeutic purpose that antibodies, Fc fusions. and the like may be used for. In a preferred embodiment, the Ep-CAM targeting proteins are administered to a patient to treat disorders including but not limited to cancer.
  • a “patient” for the purposes of the present invention includes both humans and other animals, preferably mammals and most preferably humans.
  • the Ep-CAM targeting proteins of the present invention have both human therapy and veterinary applications.
  • treatment in the present invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for a disease or disorder.
  • successful administration of an EP-CAM targeting protein prior to onset of the disease results in treatment of the disease.
  • successful administration of an optimized Ep-CAM targeting protein after clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease.
  • “Treatment” also encompasses administration of an optimized Ep-CAM targeting protein after the appearance of the disease in order to eradicate the disease.
  • Successful administration of an agent after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease, comprises treatment of the disease.
  • Those “in need of treatment” include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
  • an Ep-CAM targeting protein of the present invention is administered to a patient having a disease involving inappropriate expression of a protein or other molecule.
  • this is meant to include diseases and disorders characterized by aberrant proteins, due for example to alterations in the amount of a protein present, protein localization, posttranslational modification, conformational state, the presence of a mutant or pathogen protein, etc.
  • the disease or disorder may be characterized by alterations molecules including but not limited to polysaccharides and gangliosides.
  • An overabundance may be due to any cause, including but not limited to overexpression at the molecular level, prolonged or accumulated appearance at the site of action, or increased activity of a protein relative to normal.
  • diseases and disorders characterized by a reduction of a protein.
  • This reduction may be due to any cause, including but not limited to reduced expression at the molecular level, shortened or reduced appearance at the site of action, mutant forms of a protein, or decreased activity of a protein relative to normal.
  • Such an overabundance or reduction of a protein can be measured relative to normal expression, appearance, or activity of a protein, and the measurement may play an important role in the development and/or clinical testing of the Ep-CAM targeting proteins of the present invention.
  • cancer and “cancerous” herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
  • cancers include hematologic malignancies, such as Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphomalchronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursor cells, including B-cell acute lymphoblastic leukemiallymphoma, and T-cell acute lymphoblastic leukemiallymphoma, thymoma, tumors of the mature T and NK cells, including peripheral T-cell leukemias, adult T-cell leukemia/T-cell lymphomas and large granular lymphocytic leukemia, Langerhans cell histocytosis, myeloid neoplasias such as acute
  • lung eg. small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung
  • digestive system eg. gastric or stomach cancer including gastrointestinal cancer, cancer of the bile duct or biliary tract, colon cancer, rectal cancer, colorectal cancer, and anal carcinoma
  • reproductive system eq. testicular, penile, or prostate cancer, uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer
  • skin eg.
  • liver eg. liver cancer, hepatic carcinoma, hepatocellular cancer, and hepatoma
  • bone eq. osteoclastoma, and osteolytic bone cancers
  • additional tissues and organs eg. pancreatic cancer, bladder cancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer of the peritoneum, and Kaposi's sarcoma
  • tumors of the vascular system eg. angiosarcoma and hemagiopericytoma.
  • autoimmune diseases include allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphosphoiipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune disfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed
  • inflammatory disorders include acute respiratory distress syndrome (ARDS), acute septic arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergy, asthma, atherosclerosis, chronic inflammation due to chronic bacterial or viral infectionis, chronic obstructive pulmonary disease (COPD), coronary artery disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed hypersensitivity reactions, inflammation associated with tumors, peripherali nerve injury or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation due to meningitis, multiple organ injury syndrome, pulmonary fibrosis, sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated arthropy, and undifferentiated spondyloarthropathy.
  • ARDS acute respiratory distress syndrome
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • coronary artery disease encephalitis
  • inflammatory bowel disease inflammatory
  • infectious diseases include diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites. Infectious diseases may be caused by viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus, (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis, and the like.
  • viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes
  • Infections diseases may also be caused by bacteria including Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionelia, Helicobacter pylori, Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S. pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersinia pestis, and the like.
  • bacteria including Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionelia, Helicobacter pylori, Mycobacterium
  • Infectious diseases may also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Penicillium marneffei, and the like. Infectious diseases may also be caused by protozoa and parasites such as chlamydia, kokzidioa, leishmania, malaria, rickettsia, trypanosoma, and the like.
  • Ep-CAM targeting proteins of the present invention may be used to prevent or treat additional conditions including but not limited to heart conditions such as congestive heart failure (CHF), myocarditis and other conditions of the myocardium; skin conditions such as rosecea, acne, and eczema; bone and tooth conditions such as bone loss, osteoporosis, Paget's disease, Langerhans'cell histiocytosis, periodontal disease, disuse osteopenia, osteomalacia, monostotic fibrous dysplasia, polyostotic fibrous dysplasia, bone metastasis, bone pain management, humoral malignant hypercalcemia, periodontal reconstruction, spinal cord injury, and bone fractures; metabolic conditions such as Gaucher's disease; endocrine conditions such as Cushing's syndrome; and neurological conditions.
  • CHF congestive heart failure
  • myocarditis and other conditions of the myocardium skin conditions such as rosecea, acne, and eczema
  • bone and tooth conditions such as bone loss, osteopo
  • compositions are contemplated wherein an Ep-CAM targeting protein of the present invention and and one or more therapeutically active agents are formulated.
  • Formulations of the Ep-CAM targeting proteins of the present invention are prepared for storage by mixing the Ep-CAM targeting protein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980, expressly incorporated by reference), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidiimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the pharmaceutical composition that comprises the Ep-CAM targeting protein of the present invention may be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the formulations to be used for in vivo administration are preferrably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
  • Ep-CAM targeting proteins disclosed herein may also be formulated as immunoliposomes.
  • a liposome is a small vesicle comprising various types of lipids, phospholipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal.
  • Liposomes containing the Ep-CAM targeting protein are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et at., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. Nos. 4,485,045; 4,544,545; and PCT WO 97/38731, all expressly incorporated by reference.
  • Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556, expressly incorporated by reference.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • a chemotherapeutic agent or other therapeutically active agent is optionally contained within the liposome (Gabizon et al., 1989, J National Cancer Inst 81:1484, expressly incorporated by reference).
  • Ep-CAM targeting protein and other therapeutically active agents may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions.
  • coacervation techniques for example using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methylmethacylate) microcapsules
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the Lupron Depot® (which are injectable microspheres composed of lactic acid-glycolic acid copolymer or lactic acid polymer and leuprolide acetate), poly-D-( ⁇ )-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes, which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG)).
  • Lupron Depot® injectable microspheres composed of lactic acid-glycolic acid copolymer or lactic acid polymer and leuprolide acetate
  • ProLease® commercially available from Alkermes, which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into
  • Administration of the pharmaceutical composition comprising an Ep-CAM targeting protein of the present invention may be done in a variety of ways, including, but not limited to orally, subcutaneously, intravenously, intranasally, intraotically, transdermally, topically (eg., gels, salves, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary, vaginally, parenterally, rectally, or intraocularly.
  • the EpCAM targeting protein may be directly applied as a solution or spray.
  • the pharmaceutical composition may be formulated accordingly depending upon the manner of introduction.
  • Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition.
  • Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate (see WO 04091658).
  • Anti-Ep-CAM antibodies or Fc fusions of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
  • Ep-CAM targeting proteins of the present invention may also be delivered using such methods.
  • administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
  • Pulmonary delivery may be accomplished using an inhaler or nebulizer and a formulation comprising an aerosolizing agent.
  • AERx® inhalable technology commercially available from Aradigm, or InhanceTM pulmonary delivery system commercially available from Nektar Therapeutics may be used.
  • Ep-CAM targeting proteins of the present invention may be more amenable to intrapulmonary delivery.
  • FcRn is present in the lung, and may promote transport from the lung to the bloodstream (e.g. Syntonix WO 04004798, Bitonti et.al. (2004) Proc. Nat. Acad. Sci. 101:9763-8, both expressly incorporated by reference).
  • anti-Ep-CAM antibodes or Fc fusions that bind FcRn more effectively in the lung or that are released more efficiently in the bloodstream may have improved bioavailability following intrapulmonary administration.
  • Ep-CAM targeting proteins of the present invention may also be more amenable to intrapulmonary administration due to, for example, improved solubility or altered isoelectric point.
  • Ep-CAM targeting proteins of the present invention may be more amenable to oral delivery due to, for example, improved stability at gastric pH and increased resistance to proteolysis.
  • FcRn appears to be expressed in the intestinal epithelia of adults (Dickinson et.al. (1999) J. Clin. Invest. 104:903-11), so anti-Ep-CAM antibodies or Fc fusions of the present invention with improved FcRn interaction profiles may show enhanced bioavailability following oral administration.
  • FcRn mediated transport of Ep-CAM targeting proteins may also occur at other mucus membranes such as those in the gastrointestinal, respiratory, and genital tracts (Yoshida et. al. (2004) Immunity 20:769-83).
  • any of a number of delivery systems are known in the art and may be used to administer the Ep-CAM targeting proteins of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (eg. PLA/PGA microspheres), and the like.
  • an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used.
  • Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolic acid copolymers such as the Lupron Depot®, and poly-D-( ⁇ )-3-hydroxyburyric acid. It is also possible to administer a nucleic acid encoding the Ep-CAM targeting protein of the current invention for example by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents. In all cases, controlled release systems may be used to release the Ep-CAM targeting protein at or close to the desired location of action.
  • a nucleic acid encoding the Ep-CAM targeting protein of the current invention for example by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents.
  • controlled release systems may be used to release the Ep-CAM
  • the dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutically or prophylactically effective.
  • adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • the concentration of the therapeutically active Ep-CAM targeting protein in the formulation may vary from about 0.1 to 100 weight %. In a preferred embodiment, the concentration of the Ep-CAM targeting protein is in the range of 0.003 to 1.0 molar.
  • a therapeutically effective dose of the Ep-CAM targeting protein of the present invention may be administered.
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 10 mg/kg being preferred.
  • only a single dose of the Ep-CAM targeting protein is used. In other embodiments, multiple doses of the Ep-CAM targeting protein are administered.
  • the elapsed time between administrations may be less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.
  • the Ep-CAM targeting proteins of the present invention are administered in metronomic dosing regimes, either by continuous infusion or frequent administration without extended rest periods.
  • metronomic administration may involve dosing at constant intervals without rest periods.
  • regimens encompass chronic low-dose or continuous infusion for an extended period of time, for example 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or more.
  • the use of lower doses may minimize side effects and the need for rest periods.
  • the Ep-CAM targeting protein of the present invention and one or more other prophylactic or therapeutic agents are cyclically administered to the patient. Cycling therapy involves administration of a first agent at one time, a second agent at a second time optionally additional agents at additional times, optionally a rest period, and then repeating this sequence of administration one or more times. The number of cycles is typically from 2-10. Cycling therapy may reduce the development of resistance to one or more agents, may minimize side effects, or may improve treatment efficacy.
  • Ep-CAM targeting proteins of the present invention may be administered concomitantly with one or more other therapeutic regimens or agents.
  • the additional therapeutic regimes or agents may be used to improve the efficacy or safety of the Ep-CAM targeting protein.
  • the additional therapeutic regimes or agents may be used to treat the same disease or a comorbidity rather than to alter the action of the Ep-CAM targeting protein.
  • an Ep-CAM targeting protein of the present invention may be administered to the patient along with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.
  • Ep-CAM targeting protein of the present invention may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, additional Ep-CAM targeting proteins, Fc ⁇ RIIb or other Fc receptor inhibitors, or other therapeutic agents.
  • cytotoxic agents including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitor
  • the terms “in combination with” and “co-administration” are not limited to the administration of the prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the Ep-CAM targeting protein of the present invention and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either the Ep-CAM targeting protein of the present invention or the other agent or agents. It is preferred that the Ep-CAM targeting protein and the other agent or agents act additively, and especially preferred that they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The skilled medical practitioner can determine empirically, or by considering the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration.
  • the Ep-CAM targeting proteins of the present invention are administered with one or more additional molecules comprising antibodies or Fc.
  • the Ep-CAM targeting proteins of the present invention may be co-administered with one or more other antibodies that have efficacy in treating the same disease or an additional comorbidity; for example two antibodies may be administered that recognize two antigens that are overexpressed in a given type of cancer, or two antigens that mediate pathogenesis of an autoimmune or infectious disease.
  • anti-cancer antibodies examples include, but are not limited to, anti 17-IA cell surface antigen antibodies such as PanorexTM (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies; anti-A33 antibodies such as A33 and CDP-833; anti- ⁇ 4 ⁇ 1 integrin antibodies such as natalizumab; anti- ⁇ 4 ⁇ 7 integrin antibodies such as LDP-02; anti- ⁇ V ⁇ 1 integrin antibodies such as F-200, M-200, and SJ-749; anti- ⁇ V ⁇ 3 integrin antibodies such as aboiximab, CNTO-95, Mab-17E6, and VitaxinTM; anti-complement factor 5 (C5) antibodies such as 5G1.1; anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodies such as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such as IDEC-151, MDX-CD4, OK
  • anti-idiotype antibodies including but not limited to the GD3 epitope antibody BEC2 and the gp72 epitope antibody 105AD7, may be used.
  • bispecific antibodies including but not limited to the anti-CD3/CD20 antibody Bi20 may be used.
  • antibodies that may be co-administered to treat autoimmune or inflammatory disease, transplant rejection, GVHD, and the like include, but are not limited to, anti- ⁇ 4 ⁇ 7 integrin antibodies such as LDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement (C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322, MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD 11a antibodies, anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23 antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax, anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64 antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114, anti-CD147 antibodies such as ABX-CBL, anti-E-select
  • Fc-containing molecules that may be co-administered to treat autoimmune or inflammatory disease, transplant rejection, GVHD, and the like include, but are not limited to, the p75 TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.
  • antibodies that may be co-administered to treat infectious diseases include, but are not limited to, anti-anthrax antibodies such as ABthrax, anti-CMV antibodies such as CytoGam and sevirumab, anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G, anti-helicobacter antibodies such as Pyloran, anti-hepatitis B antibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such as HRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab, RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb, BSYX-A110, and SE-Mab.
  • anti-anthrax antibodies such as ABthrax
  • anti-CMV antibodies such as CytoGam and sevirumab
  • anti-cryptosporidium antibodies such as CryptoGAM
  • Sporidin-G anti-helicobacter antibodies
  • anti-helicobacter antibodies such as Py
  • the Ep-CAM targeting proteins of the present invention may be co-administered with one or more other molecules that compete for binding to one or more Fc receptors.
  • co-administering inhibitors of the inhibitory receptor Fc ⁇ RIIb may result in increased effector function.
  • co-administering inhibitors of the activating receptors such as Fc ⁇ RIIIa may minimize unwanted effector function.
  • Fc receptor inhibitors include, but are not limited to, Fc molecules that are engineered to act as competitive inhibitors for binding to Fc ⁇ RIIb Fc ⁇ RIIIa, or other Fc receptors, as well as other immunoglobulins and specificially the treatment called IVIg (intravenous immunoglobulin).
  • the inhibitor is administered and allowed to act before the Ep-CAM targeting protein is administered.
  • An alternative way of achieving the effect of sequential dosing would be to provide an immediate release dosage form of the Fc receptor inhibitor and then a sustained release formulation of the Ep-CAM targeting protein of the invention.
  • the immediate release and controlled release formulations could be administered separately or be combined into one unit dosage form.
  • Administration of an Fc ⁇ RIIb inhibitor may also be used to limit unwanted immune responses, for example anti-Factor VIII antibody response following Factor VIII administration to hemophiliacs.
  • the Ep-CAM targeting proteins of the present invention are administered with a chemotherapeutic agent.
  • chemotherapeutic agent as used herein is meant a chemical compound useful in the treatment of cancer.
  • alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM), alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; antibiotics such as aclacinomysins, actinomycin, authramycin, aza
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (such as Tomudex), additional chemotherapeutics including aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; difluoromethylornithine (DMFO); elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mritoxantrone, mopidamol; nitracrine; pentostatin; phena
  • a chemotherapeutic or other cytotoxic agent may be administered as a prodrug
  • prodrug as used herein is meant a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, for example Wilman, 1986, Biochemical Society Transactions, 615th Meeting Harbor, 14:375-382; and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery, ” Directed Drug Delivery, Borchardt et al. (ed.): 247-267, Humana Press, 1985; both expressly incorporated by reference.
  • the prodrugs that may find use with the present invention include but are not limited to phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • Examples of cytotoxic drugs that can be derivatized into a prodrug form for use with the Ep-CAM targeting proteins of the present invention include but are not limited to any of the aforementioned chemotherapeutic agents.
  • the Ep-CAM targeting protein is administered with an anti-angiogenic agent.
  • anti-angiogenic agent as used herein is meant a compound that blocks, or interferes to some degree, the development of blood vessels
  • the anti-angiogenic factor may, for instance, be a small molecule or a protein, for example an antibody, Fc fusion, or cytokine, which binds to a growth factor or growth factor receptor involved in promoting angiogenesis.
  • the preferred anti-angiogenic factor herein is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).
  • VEGF Vascular Endothelial Growth Factor
  • RNA-based therapeutics that reduce levels of VEGF or VEGF-R expression, VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bind VEGF-R.
  • the Ep-CAM targeting protein is administered with a therapeutic agent that induces or enhances adaptive immune response, for example an antibody that targets CTLA-4.
  • Additional anti-angiogenesis agents include, but are not limited to, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin, bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), farnesyl transferase inhibitors, fibronectin fragment gro-beta, halofuginone, heparinases, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon inducible protein 10 (IP-10), interleukin-12, kringle 5 (plasminogen fragment), marimastat.
  • angiostatin plasminogen fragment
  • TIMPs metalloproteinase inhibitors
  • 2-methodyestradiol MMI 270 (CGS 27023A), plasminogen activiator inhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16kDa fragment, proliferin-related protein (PRP), PTK 787/ZK 222594, retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-470, transforming growth factor beta (TGF- ⁇ ), vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.
  • TIMPs transforming growth factor beta
  • TGF- ⁇ transforming growth factor beta
  • vasculostatin vasostatin (calreticulin fragment),
  • the Ep-CAM targeting protein is administered with a tyrosine kinase inhibitor.
  • tyrosine kinase inhibitor as used herein is meant a molecule that inhibits to some extent tyrosine kinase activity of a tyrosine kinase.
  • inhibitors include but are not limited to quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g.
  • the EP-CAM targeting protein is administered with one or more immunomodulatory agents.
  • immunomodulatory agents may increase or decrease production of one or more cytokines, up- or down-regulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells.
  • Immunomodulatory agents include but not limited to: non-steroidal anti-inflammatory drugs (NSAIDs) such as asprin, ibuprofed, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (eg.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • glucocorticoids dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidineicosanoids such as prostaglandins, thromboxanes, and leukotrienes; as well as topical steroids such as anthralin, calcipotriene, clobetasol, and tazarotene); cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine, chemokine, or receptor antagonists including antibodies, soluble receptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCRS, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64,
  • Ep-CAM targeting protein of the present invention are administered with a cytokine.
  • cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, insulin-like growth factor-I and -II; erythropoietin (EPO)
  • cytokines or other agents that stimulate cells of the immune system are co-administered with the Ep-CAM targeting protein of the present invention.
  • a mode of treatment may enhance desired effector function.
  • agents that stimulate NK cells including but not limited to IL-2 may be co-administered
  • agents that stimulate macrophages including but not limited to C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et. al. (2003) Scand. J. Immunol, 57: 221-8, expressly incorporated by reference), may be co-administered.
  • agents that stimulate neutrophils including but not limited to G-CSF, GM-CSF, and the like may be administered.
  • agents that promote migration of such immunostimulatory cytokines may be used.
  • additional agents including but not limited to interferon gamma, IL-3 and IL-7 may promote one or more effector functions.
  • cytokines or other agents that inhibit effector cell function are co-administered with the Ep-CAM targeting protein of the present invention.
  • Such a mode of treatment may limit unwanted effector function
  • the Ep-CAM targeting protein is administered with one or more antibiotics, including but not limited to. aminoglycoside antibiotics (eg. apramycin, arbekacin, bambermycins, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomycin, spectrinomycin), aminocyclitols (eg. sprctinomycin), amphenicol antibiotics (eg. azidamfenicol, chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics (eg.
  • aminoglycoside antibiotics eg. apramycin, arbekacin, bambermycins, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, siso
  • rifamide and rifampin carbapenems (eg. imipenem, meropenem, panipenem); cephalosporins (eg. cefaclor, cefadroxii, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine), cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan); lincosamides (eg.
  • clindamycin, lincomycin macrolide (eg, azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, tobramycin), monobactams (eg. aztreonam, carumonam, and tigernonam); mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam); penicillins (eg.
  • bacitracin colistin, polymixin B, teicoplanin, vancomycin
  • quinolones amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, rosoxacin, rufloxacin, sparfioxacin, temafloxacin, tosufloxacin, trovafloxacin); rifampin; streptogramins (eg.
  • quinupristin, dalfopristin quinupristin, dalfopristin
  • sulfonamides sulfanilamide, sulfamethoxazole
  • tetracyclenes chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, duramycin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, vancomycin).
  • Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, niconazole, nystatin, terbinafine, terconazole, and tioconazole may also be used.
  • Antiviral agents including protease inhibitors, reverse transcriptase inhibitors, and others, including type I interferons, viral fusion inhibitors, and neuramidase inhibitors, may also be used.
  • antiviral agents include, but are not limited to, acyclovir, adefovir, amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet, gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine, may be used.
  • the Ep-CAM targeting proteins of the present invention may be combined with other therapeutic regimens.
  • the patient to be treated with an anti-Ep-CAM antibody or Fc fusion of the present invention may also receive radiation therapy.
  • Radiation therapy can be administered according to protocols commonly employed in the art and known to the skilled artisan. Such therapy includes but is not limited to cesium, iridium, iodine, or cobalt radiation.
  • the radiation therapy may be whole body irradiation, or may be directed locally to a specific site or tissue in or on the body, such as the lung, bladder, or prostate.
  • radiation therapy is administered in pulses over a period of time from about 1 to 2 weeks. The radiation therapy may, however, be administered over longer periods of time.
  • radiation therapy may be administered to patients having head and neck cancer for about 6 to about 7 weeks.
  • the radiation therapy may be administered as a single dose or as multiple, sequential doses.
  • the skilled medical practitioner can determine empirically the appropriate dose or doses of radiation therapy useful herein.
  • the Ep-CAM targeting protein of the present invention and one or more other anti-cancer therapies are employed to treat cancer cells ex vivo. It is contemplated that such ex vivo treatment may be useful in bone marrow transplantation and particularly, autologous bone marrow transplantation.
  • treatment of cells or tissue(s) containing cancer cells with Ep-CAM targeting protein and one or more other anti-cancer therapies, such as described above can be employed to deplete or substantially deplete the cancer cells prior to transplantation in a recipient patient.
  • Radiation therapy may also comprise treatment with an isotopically labeled molecule, such as an antibody.
  • an isotopically labeled molecule such as an antibody.
  • radioimmunotherapeutics include but ZevalinTM (Y-90 labeled anti-CD20), LymphoCideTM (Y-90 labeled anti-CD22) and BexxarTM (1-131 labeled anti-CD20)
  • Ep-CAM targeting proteins of the invention may employ in combination with still other therapeutic techniques such as surgery or phototherapy.
  • a number of the receptors that may interact with the Ep-CAM targeting proteins of the present invention are polymorphic in the human population.
  • the efficacy of the Ep-CAM targeting proteins of the present invention may be affected by the presence or absence of specific polymorphisms in proteins.
  • Fc ⁇ RIIIA is polymorphic at position 158, which is commonly either V (high affinity) or F (low affinity).
  • Patients with the V/V homozygous genotype are observed to have a better clinical response to treatment with the anti-CD20 antibody Rituxan® (rituximab), likely because these patients mount a stronger NK response (Dall'Ozzo et. al. (2004) Cancer Res. 64:4664-9, expressly incorporated by reference).
  • Ep-CAM targeting proteins of the present invention may bind preferentially to a particular polymorphic form of a receptor, for example Fc ⁇ RIIIA 158 V, or to bind with equivalent affinity to all of the polymorphisms at a particular position in the receptor, for example both the 158V and 158F polymorphisms of Fc ⁇ RIIIA.
  • Ep-CAM targeting proteins of the present invention may have equivalent binding to polymorphisms may be used in an antibody to eliminate the differential efficacy seen in patients with different polymorphisms.
  • Such a property may give greater consistency in therapeutic response and reduce non-responding patient populations.
  • Such variant Fc with identical binding to receptor polymorphisms may have increased biological activity, such as ADCC, CDC or circulating half-life, or alternatively decreased activity, via modulation of the binding to the relevant Fc receptors.
  • Ep-CAM targeting proteins of the present invention may bind with higher or lower affinity to one of the polymorphisms of a receptor, either accentuating the existing difference in binding or reversing the difference.
  • Such a property may allow creation of therapeutics particularly tailored for efficacy with a patient population possessing such polymorphism.
  • a patient population possessing a polymorphism with a higher affinity for an inhibitory receptor such as Fc ⁇ RIIB could receive a drug containing an Ep-CAM targeting protein with reduced binding to such polymorphic form of the receptor, creating a more efficacious drug,
  • patients are screened for one or more polymorphisms in order to predict the efficacy of the Ep-CAM targeting proteins of the present invention.
  • This information may be used, for example, to select patients to include or exclude from clinical trials or, post-approval, to provide guidance to physicians and patients regarding appropriate dosages and treatment options,
  • Fc ⁇ RIIIA 158F antibody drugs such as the anti-CD20 mAb
  • Rituximab are minimially effective (Carton 2002 Blood 99: 754-758; Weng 2003 J. Clin. Oncol. 21:3940-3947); such patients may show a much better clinical response to the antibodies of the present invention.
  • patients are selected for inclusion in clinical trials for an antibody of the present invention if their genotype indicates that they are likely to respond significantly better to an antibody of the present invention as compared to one or more currently used antibody therapeutics.
  • appropriate dosages and treatment regimens are determined using such genotype information.
  • patients are selected for inclusion in a clinical trial or for receipt of therapy post-approval based on their polymorphism genotype, where such therapy contains an Ep-CAM targeting protein engineered to be specifically efficacious for such population, or alternatively where such therapy contains an Ep-CAM targeting protein that does not show differential activity to the different forms of the polymorphism.
  • Included in the present invention are diagnostic tests to identify patients who are likely to show a favorable clinical response to an Ep-CAM targeting protein of the present invention, or who are likely to exhibit a significantly better response when treated with an Ep-CAM targeting protein of the present invention versus one or more currently used antibody therapeutics. Any of a number of methods for determining Fc ⁇ R polymorphisms in humans known in the art may be used.
  • the present invention comprises prognostic tests performed on clinical samples such as blood and tissue samples. Such tests may assay for effector function activity, including but not limited to ADCC, CDC, phagocytosis, and opsonization, or for killing, regardless of mechanism, of cancerous or otherwise pathogenic cells.
  • ADCC assays such as those described previously, are used to predict, for a specific patient, the efficacy of a given Ep-CAM targeting protein of the present invention. Such information may be used to identify patients for inclusion or exclusion in clinical trials, or to inform decisions regarding appropriate dosages and treatment regimens. Such information may also be used to select a drug that contains a particular Ep-CAM targeting protein that shows superior activity in such assay.
  • FIGS. 1 and 2 provide some heavy and light chain variable region sequences of the anti-Ep-CAM antibodies used in the present study.
  • the mouse, parent chimeric heavy and light chains are labeled H0 17-1A and L0 17-1A respectively. Due to the wide use of hybridoma technology, a substantial number of antibodies are derived from nonhuman sources. However, nonhuman proteins are often immunogenic when administered to humans, thereby greatly reducing their therapeutic utility. Immunogenicity is the result of a complex series of responses to a substance that is perceived as foreign, and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis.
  • Efficacy can be reduced directly by the formation of neutralizing antibodies. Efficacy may also be reduced indirectly, as binding to either neutralizing or non-neutralizing antibodies typically leads to rapid clearance from serum. Severe side effects and even death may occur when an immune reaction is raised.
  • protein engineering is used to reduce the immunogenicity of the Ep-CAM targeting proteins of the present invention.
  • the immunogenicity of the anti-Ep-CAM antibody 17-1A was reduced using a method described in U.S. Ser. No. 60/619,483, filed Oct. 14, 2004 and U.S. Ser. No. 11/004,590, entitled “Methods of Generating Variant Proteins with Increased Host String Content and Compositions Thereof”, filed on Dec. 6, 2004.
  • the methods reduce the potential for immunogenicity by increasing the human string content of the antibody through mutations.
  • the heavy and light chains with reduced potential for immunogenicity are named H1, H2, H3, H4, H5, H6, H2.1, H2.2, etc and L1, L2, L3, L4, L3.1, L3.2 etc and are shown in FIGS. 1 and 2 .
  • the heavy and light chains of the original antibody, 17-1A are referred to as H0 and L0.
  • Anti-Ep-CAM antibodies were expressed by transient transfection of vectors encoding the heavy and light chains into 293T cells grown in 10% ultra low IgG fetal bovine serum with 1 rmM sodium pyruvate and 1 ⁇ non-essential amino acids (Gibco®, Invitrogen Hayward Calif.). Five days after transfection, the culture media was removed and ran through a protein A column (Pierce Biotechnology Inc, Rockford Md.).
  • FIG. 4 shows typical yields of some Ep-CAM-binding proteins.
  • the heavy chains may be made with any type of constant domain including, in humans, IgG1, IgG2 and hybrids comprising IgG1 and IgG2 as well as mouse constant domains such as IgG1 and IgG2a, which may be referred to as mIgG1 and mIgG2a.
  • the sequences of many of these heavy chains may be found in FIG. 3 .
  • Data demonstrating the use of the hybrid IgG1, IgG2 heavy chain may be found in FIGS. 4, 5 and 20 .
  • FIG. 7 shows a schematic representation of the AlphaScreen assay. Binding affinity of anti-Ep-CAM antibodies to the extracellular domain of Ep-CAM was measured using a quantitative and sensitive method, AlphaScreenTM assay.
  • the AlphaScreenTM assay is a bead-based non-radioactive luminescent proximity assay. Laser excitation of a donor bead excites oxygen, which if sufficiently close to the acceptor bead will generate a cascade of chemiluminescent events, ultimately leading to fluorescence emission at 520-620 nm.
  • the AlphaScreenTM assay was applied as a competition assay for screening the antibodies.
  • Wild-type IgG1 Ep-CAM antibody was biotinylated and extracellular domain of Ep-CAM was DIGylated by standard methods for attachment to streptavidin donor beads and anti-DIG acceptor beads. In the absence of competing anti-Ep-CAM antibodies (unlabeled), wild-type antibody (biotinylated) and Ep-CAM (DIGylated) interact and produce a signal at 520-620 nm. Addition of untagged antibody competes with wild-type biotinylated anti-Ep-CAM and DIGylated-Ep-CAM interaction reducing fluorescence quantitatively to enable determination of relative binding affinities.
  • FIGS. 8 to 3 show representative AlphaScreenTM data of various humanized antibodies of the present invention. These figure show competition AlphaScreenTM data in which tested antibody in this case, competes with a reference antibody for the binding to Ep-CAM or an antibody binding protein. The binding of the humanized antibodies for the antigen, Ep-CAM, and protein A are shown on the left and right sides, respectively, of the FIG. 8 . The results are also summarized in FIG. 9 . Most humanized antibodies have an Ep-CAM binding affinity within 2-fold of the wild type. That is, they have between 0.5 and 2.0 fold increases in binding affinity relative to the wild type. Fold increase values greater than 1.0 demonstrate stronger binding than the wild type, 17-1A. FIGS. 10 to 13 show repeat measurements of the humanized antibodies using the AlphaScreenTM method.
  • FIGS. 14 to 16 show binding reactions for WT and variant anti-Ep-CAM antibodies measured with surface plasmon resonance.
  • the kinetic constants for the binding of anti-Ep-CAM antibodies to Ep-CAM antigen were determined using surface plasmon resonance-based measurements on a BIAcore 3000 instrument (Biacore, Uppsala, Sweden).
  • the second and fourth CM5 sensor chip flow cells were coupled with recombinant human Ep-CAM/Fc (R&D Systems, Minneapolis, Minn.) using amine chemistry. Approximately 1000 and 6000 response units were respectively immobilized.
  • the first and third flow cells were ethanolamine blocked to serve as reference flow cells.
  • Binding experiments were performed by injecting antibodies over the Ep-CAM/Fc and reference flow cells at varying concentrations ranging from 1 nM to 1000 nM in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20, filtered & degassed (HBS-EP, Biacore, Uppsala, Sweden) using KINJECT (2 minutes association, 2 minutes dissociation) at 50 ⁇ l/minute. The sensor chip was regenerated with 10 mM glycine, pH 1.5. Data transformation was prepared by subtraction of blank injections (buffer without analyte) and y-transform prior to the injection start to zero using BlAevaluation software.
  • the responses between 12.5 and 100 nM were globally analyzed using a one to one interaction (Langmuir) binding model.
  • the corresponding association rate constant (k a ), dissociation rate constant (k d ), maximum analyte binding capacity (R max ), bulk refractive index contribution (RI), equilibrium association constant (K A ), equilibrium dissociation constant (K D ), steady state binding level (R eq ), and observed rate constant (k obs ) derived from these fits are presented in FIG. 14 , along with the chi-square (X 2 ) value which represents the closeness of fit between the fit curve and the actual data curve.
  • the data for the variants in FIG. 15 was collected similarly, but the resulting binding/dissociation curves were not fit to a particular binding model.
  • the binding strength is presented as the SPR signal, the response units, after a fixed time of flowing the analyte over the surface. Stronger binding interactions yield a higher number of response units after this fixed time.
  • the SPR association/dissociation curves of three variants are shown in FIG. 16 .
  • Three different concentrations are shown for each variant and over-laid onto the data curves are fitted curve generated with a 1:1 binding model containing a drifting baseline.
  • the three curves for each variant were fit simultaneously to the model yielding one dissociation constant describing the binding of the variant to Ep-CAM.
  • the variant H3.77/L3 showed a dissociation constant of 6.49e-8 M ⁇ 1 in this assay.
  • FIG. 17 shows the binding of a humanized anti-Ep-CAM and trastuzumab to the gastric carcinoma line, KATOIII, and the breast cancer line, SkBr3.
  • Each cell line was dissociated using Accutase wash, resuspended and seeded at 50,000 cells per well of a 96-well plate.
  • Cells were either treated with a secondary antibody-fluor (PE) conjugate or first treated with either trastuzumab or anti-Ep-CAM followed by secondary mAb treatment.
  • PE secondary antibody-fluor
  • ADCC was measured using either the DELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer) or LDH Cytotoxicity Detection Kit (Roche Diagnostic).
  • Human PBMCs were purified from leukopacks using a ficoll gradient.
  • NK cells were isolated from human PBMCs using negative selection and magnetic beads (Miltenyi Biotec).
  • target cells were first loaded with BATDA at 1 ⁇ 10 6 cells/ml and washed 4 times.
  • target cells were seeded into 96-well plates at 10,000 cells/well, and opsonized using antibodies at the indicated final concentration. Triton X100 and PBMCs alone were typically run as positive and negative controls.
  • Effector cells were added at 25:1 PBMCs:target cells or 4:1 NK Cells:target cells, and the plate was incubated at 37° C. for 4 hrs. Cells were incubated with either Eu3+ solution or LDH reaction mixture, and fluorescence was measured using the Fusion Alpha-FP. Data were normalized to maximal (triton) and minimal (PBMCs alone) lysis, and fit to a sigmoidal dose-response model.
  • FIGS. 18 to 24 display the results of ADCC assays of various anti-Ep-CAM antibodies. Improved, ie greater, levels of ADCC may be seen as either a shift in potency or efficacy. Improved potency of antibody is seen as a left shift of an ADCC curve compared to a reference curve. The left shift indicates that less antibody is required to achieve the same degree of cytotoxicy as the reference antibody. In addition, improved ADCC may also be evident as improved efficacy, which is seen as an upward shift in the ADCC curve compared to a reference curve. The upward shift indicates that the same amount of antibody produces a greater degree of cytotoxicity. Improvements in potency and efficacy may occur simultaneously or separately depending on the two antibodies being compared, the assay conditions (cell lines used, antibody concentrations, etc) and other factors.
  • the humanized anti-Ep-CAM antibody H3.77_L3 WT may be used as a reference, wild-type, antibody.
  • This antibody also shows an increase in efficacy, because at log antibody concentration of 1.0, it has about 30% cytoxicity whereas the reference antibody has about 20% cytotoxicity.
  • H3.77_L3 G236A shows increased efficacy, but very little change in potency as it shows more cytoxicity at higher antibody concentrations, but very little change in the midpoint of its ADCC curve. Also indicated in FIG. 20 a is the improved potency of H3.77_L3 S239D/I332E compared to H3.77_L3 G236A with very little change in efficacy. Relative to the H3.77_L3 WT antibody, all of the other antibodies shown in FIG. 20 a have improved efficacy, improved potency, or an improvement in both efficacy and potency.
  • Ep-CAM-binding antibodies may comprise substitutions in the Fc region, or other regions, to optimize the antibody function.
  • FIGS. 4, 5 , 13 , and 20 to 23 comprise data of anti-Ep-CAM antibodies comprising substitutions in the Fc domain.
  • the substitutions S239D, I332E, G326A, L235G, G236R, A330Y, H268E affect binding to the Fcgamma receptors (See U.S. ser. No. 11/124620 entitled “Optimized Fc Variants”).
  • the substitutions P257L, P257N, V308F, V308Y, V279Y, and Q311 V affect binding to FcRn (See PCT WO06053301A2 entitled “Fc Variants with altered binding to FCRN”).
  • the altered Fc receptor binding and effector function (ADCC) of many variants are shown in FIGS. 13 and 20 to 23 .
  • Many anti-Ep-CAM antibodies were found to have increased killing of LS180 and HT29 cells, particularly those that comprise the modifications S239D, I332E, G326A, L235G, G236R, A330Y, or H268E.
  • the increased ADCC of these variants may be made in human or mouse Fc regions, including human IgG1, and hybrids of two different human IgG's, as shown for example in FIG. 13 .
  • the optimal anti-Ep-CAM clinical candidate may comprise an altered glycoform.
  • An Ep-CAM binding protein was expressed in Lec13 cells and purified by the standard methods described herein, including protein A chromatography.
  • This Lec 13 expressed antibody is a glycoform variant in that it is defucosylated; it lacks the fucose residue on it N-linked carbohydrate moiety connected to Asn297.
  • the purified protein is shown in FIG. 6 and shows the expected molecular weights of the heavy and light chains.
  • This defucosylated anti-Ep-CAM has stronger binding to the Fc receptor, FcgammaRIIIa (Val variant), as shown in FIG. 25 , lower panel.
  • the Kd measurement were made with Surface Plasmon Resonance fixing the antibody on the surface and flowing FcgammaRIIIA over the chip.
  • phagocytosis by monocytes and macrophages may be initiated by antibody binding to Fc ⁇ RIIa (Hunter et al. Blood 91(5):1762-1768, Tridandapani et al 2002 Journal of Biological Chemistry 277(7):5082-5089).
  • Fc ⁇ RIIb may also bind the antibodies but Fc ⁇ RIIb does not induce phagocytosis by monocytes and macrophages. Therefore, Fc ⁇ RIIb may passively inhibit phagocytosis by binding antibody that otherwise may be available to bind to Fc ⁇ RIIA as well under go its more active inhibitory functions.
  • Fc ⁇ RIIIa is the important Fc receptor for causing activation of NK cells.
  • Fc ⁇ RIIa and Fc ⁇ RIIb are expressed on monocytes, macrophages, neutrophils, and dendritic cells, and some of these cell types are also known to express Fc ⁇ RIIIa. It is well known in the art that activation of these cell types can depend on the relative expression and/or activation of Fc ⁇ RIIa compared to Fc ⁇ RIIb, and that coactivation of Fc ⁇ RIIb with Fc ⁇ RIIa can decrease the activation via Fc ⁇ RIIa. We therefore determined the affinity of several Fc modified Ep-CAM-targeting antibodies to several human FcR's.
  • the Kd values for a series of Fc modified Ep-CAM-targeting antibodies are shown in FIGS. 26 and 27 .
  • the affinity values show several important trends.
  • variants containing the substitutions S239D, I332E and H268E all have increased affinity for Fc ⁇ RIIIa relative to the wt IgG1 control.
  • substitutions, individually or in combinations have “Fold KD” ( FIG. 27 ) values greater than one.
  • H3.77_L3 S239D IgG1 has a Fc ⁇ RIIIa Fold KD value of 5.6, demonstrating that it has 5.6-fold stronger binding to Fc ⁇ RIIIa than the wild type.
  • Antibodies with these modifications also have increase affinity for the Fc receptors Fc ⁇ RIIa and Fc ⁇ RIIb.
  • variants containing the G236A substitutions are variants containing the G236A substitutions. All of these variants have specifically enhanced affinity for Fc ⁇ RIIa. G236A results in a specific enhancement of Fc ⁇ RIIa binding compared to ScyRIIb binding. Indeed, the RIIa/RIIb affinity ratio of G236A-containing variants is systematically improved, having a ⁇ log(RIIa/RIIB) value of about 1.0. This value means that the variants have about a full log, or 10-fold, increased binding for Fc ⁇ RIIa compared to Fc ⁇ RIIb. These variants will find utility in treatment of Ep-CAM expressing cancers, where monocytes, macrophages, neutrophils, and dendritic cells are important effector cells.
  • FIGS. 26 and 27 show data collected with antibodies comprising either the human IgG1 or a hybrid Fc comprising both IgG1 and IgG2 sequences.
  • FIG. 3 shows the sequences of some Fc domains used herein.
  • IgG allotypes may be used as Fc domains in an Ep-CAM-targeting protein.
  • Gm polymorphism is determined by the IGH1, IGH2, and IGH3 genes, which have alleles encoding allotypic antigenic determinants referred to as G1m, G2m, and G3m allotypes for markers of the human IgG1, IgG2 and IgG3 molecules.
  • FIG. 28 a provides some common allotypes, as is well known in the art. One or more of these allotypic mutations could be made in either the IgG1 or hybrid Ep-CAM-targeting antibodies by incorporating substitution, as illustrated in FIG. 28 b.

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US20070161783A1 (en) 2007-07-12
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WO2007008943A2 (fr) 2007-01-18
EP2004695A2 (fr) 2008-12-24
US7557190B2 (en) 2009-07-07

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