WO2013164689A2 - Humanized pan-her antibody compositions - Google Patents

Humanized pan-her antibody compositions Download PDF

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Publication number
WO2013164689A2
WO2013164689A2 PCT/IB2013/001027 IB2013001027W WO2013164689A2 WO 2013164689 A2 WO2013164689 A2 WO 2013164689A2 IB 2013001027 W IB2013001027 W IB 2013001027W WO 2013164689 A2 WO2013164689 A2 WO 2013164689A2
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WIPO (PCT)
Prior art keywords
antibody
seq
variable region
egfr
chain variable
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PCT/IB2013/001027
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French (fr)
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WO2013164689A8 (en
WO2013164689A3 (en
Inventor
Kim Vilbour Andersen
Peter Sejr ANDERSEN
Magnus Strandh
Klaus Koefoed
Lars Sogaard NIELSEN
Mikkel Wandahl Pedersen
Helle Jacobsen
Michael Kragh
Ida KJAER
Thomas Tuxen POULSEN
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Lantto, Johan
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Priority to MX2014013213A priority Critical patent/MX358728B/en
Priority to KR1020147033605A priority patent/KR101933990B1/en
Application filed by Lantto, Johan filed Critical Lantto, Johan
Priority to CA2872226A priority patent/CA2872226A1/en
Priority to BR112014027291A priority patent/BR112014027291A2/en
Priority to EP13729074.8A priority patent/EP2844675B1/en
Priority to JP2015509512A priority patent/JP6325527B2/en
Priority to AU2013255537A priority patent/AU2013255537B2/en
Priority to CN201380033627.0A priority patent/CN104428318B/en
Priority to US14/398,441 priority patent/US9527913B2/en
Publication of WO2013164689A2 publication Critical patent/WO2013164689A2/en
Publication of WO2013164689A3 publication Critical patent/WO2013164689A3/en
Publication of WO2013164689A8 publication Critical patent/WO2013164689A8/en
Priority to IL235386A priority patent/IL235386A0/en
Priority to HK15103006.9A priority patent/HK1202431A1/en
Priority to US15/350,801 priority patent/US10058610B2/en
Priority to US16/058,086 priority patent/US20180344846A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6859—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from liver or pancreas cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
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    • 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
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to novel humanized recombinant antibodies targeting the epidermal growth factor receptor (EGFR) family and compositions comprising two or more of these antibodies for use in cancer therapy.
  • EGFR epidermal growth factor receptor
  • the epidermal growth factor receptor family (EGFR or ErbB/HER family) is a subgroup of the receptor tyrosine kinases (RTKs) and includes four members: EGFR/ErbB, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4.
  • the members of the EGFR family are closely related single- chain modular glycoproteins with an extracellula ligand binding region, a single
  • EGFR, HER2 and HER3 are believed to play crucial roles in the malignant transformation of normal ceils and in the continued growth of cancer cells.
  • EGFR and HER2 have been found to be overexpressed by many epithelial cancers. Overexpression of EGFR and HER2 has furthermore been linked to disease progression, reduced survival, poor response and chemotherapy resistance in several human epithelial cancers. The role of HER4 in malignant transformation and cancer progression is controversial and will not be discussed further here.
  • EGFR and HER2 are validated cancer targets and both monoclonal antibodies and small molecule inhibitors of their tyrosine kinase have been approved for the treatment of various cancers.
  • HERS is currently being explored as a potential therapeutic target.
  • patients who initially respond to these therapies often relapse due to evolvement of acquired resistance.
  • Pre-c!inicai research points to the involvement of the one or both of the non- targeted receptors in the development of resistance.
  • the ErbB receptors have the ability to replace one another in order to maintain growth stimulatory signaling and a malignant phenotype. Simultaneous targeting of two or ail three receptors could therefore be a more efficient way of inhibiting cancer cells with ErbB family dependency.
  • EGFR is a 170kDa ceil surface glycoprotein consisting of a single poiypeptide chain of 1136 amino acid residues as originally determined and described by cloning and sequencing of human cD!MAs from a human vulval carcinoma ceil line.
  • EGFR contains three major domains: an extracellular domain, a transmembrane domain and an intracellular domain containing the tyrosine kinase.
  • the catalytic activity of EGFR resides in the tyrosine kinase domain (residues 685-953) and is activated upon iigand binding .
  • the EGFR exists in two different conformations, namely a tethered conformation (closed) and an extended conformation (open) .
  • the receptor shifts between the two conformations.
  • domains II and IV of the extracellular region of EGFR interact, leaving the receptor in an autoinhibited state.
  • domain III is held at a significant distance from domain I, whereby binding of EGF to both domains simultaneously is impossible.
  • domains I, II and III are stericaliy arranged in a C shape, giving room for EGF binding .
  • the conformational changes induce exposure of a ⁇ - hairpin consisting of a 20 residue region in domain II, also known as the "dimerization arm".
  • the dimerization arm extending from domain II of the EGFR makes extensive contacts with the domain II of another EGFR, thereby forming an EGFR homodimer.
  • Dimerization brings the active cytoplasmic tyrosine kinase domains of the receptors close enough for phosphorylation of the tyrosine residues in the regulatory regions of the receptors. Furthermore, the juxtamembrane regions of the two receptors form an antiparaliel dimer which has been found to be important in stabilizing the tyrosine kinase dimer.
  • the "receptor- mediated" dimerization mechanism is unique for the ErbB family compared to other tyrosine kinase receptors where "ligand-mediated" dimerization is the more common theme .
  • tyrosine kinase domain of EGFR A number of modes of activation of the intracellular tyrosine kinase domain of EGFR have been suggested .
  • the EGFR tyrosine kinase domain by default adopts a conformation normally observed only in phosphorylated and activated kinases. This indicates that the kinase domain of EGFR is constitutiveiy active. Regulation of a constitutive tyrosine kinase would thus occur through the delivery of a dimerization partner's C-terminal regulator region for trans-phosphoryiation.
  • activation of the tyrosine kinase domain involves displacement of inhibitory interactions that have not been visualized in crystallographic studies.
  • the clathrin-mediated endocytosis is the most important mechanism of down-regulation of EGFR,
  • the destiny of EGFR depends on the stabiiity of the ligand-receptor complex.
  • the EGFR homodimer Upon EGF binding to EGFR the EGFR homodimer is rapidly targeted to clatbrin-coated pits and internalized through !igand-induced endocytosis.
  • EGFR is heavily ubiquitinated by the attachment of both monoubiquitin and poiyubiquitin.
  • the ubiquitin iigase Cbi is responsible for the ubiquitination of EGFR.
  • Cbi binds either directly or indirectly through an adaptor protein such as Grb2 to phosphorylated tyrosine residues at the regulatory region of EGFR.
  • the binding of Cbi to EGFR via Grb2 is necessary for receptor internalization.
  • EsplS also plays a role in EGFR internalization. The exact role of Espl5 is however still controversial.
  • the ubiquitination is involved in endocytotic downregulation of EGFR and endosomal sorting of EGFR to lysosomes.
  • the ubiquitin chains are recognized by the endosomal sorting complex required for transport (ESCRT) and the Hrs/STA , which retains ubiquinated proteins in the membrane of early endosomes, thereby hindering recycling of EGFR.
  • EGFR is sorted into intra luminal vesicles (ILVs), which leads to delivery of EGFR to the late endosome and finally degradation In the lysosomes.
  • IMVs intra
  • TGF-a binding In contrast to the degradation of EGFR when bound to EGF, TGF-a binding allows receptor recycling.
  • the TGF-a iigand dissociates rapidly from EGFR in the early endosome due to the acidic environment, leading to receptor dephosphorylation, de-ubiquitination and thereby recycling of the receptor back to the cell surface.
  • HER2 Human epidermal growth factor receptor 2 (HER2, ErbB2 or Neu) was first described in 1984 by Schechter et ai.
  • HER2 consists of 1234 amino acids and is structurally similar to EGFR with an extracellular domain consisting of four subdomains I-IV, a transmembrane domain, a juxtamembrane domain, an Intracellular cytoplasmic tyrosine kinase and a regulatory C- termina! domain.
  • HER2 The domain Il-iV contact that restricts the domain arrangement in the tethered EGFR is absent in HER2.
  • Three of the seven conserved residues important for stabilizing the tether in the unactivated EGFR are different in HER2.
  • HER2 thus resembles EGFR in its extended (open) form with the dimerization arm exposed and apparently poised to drive receptor-receptor interactions.
  • the absence of a tethered HER2 conformation indicates that the receptor lacks autoinhibition as seen for the other members of the ErbB family.
  • a stable interface of subdomain T-III seems to keep HER2 in the extended configuration similar to the extended configuration of the EGFR-EGF complex.
  • domains I and ⁇ The interaction between domains I and ⁇ involves regions corresponding to ligand-binding sites in domains ⁇ and ⁇ of EGFR, leaving no space stericaily for ligands, rendering HER2 Incapable of binding ligands. Domains II and IV form two distinct interfaces that stabilize the heterodimer formation of HER2 and another member of the ErbB family. Biophysical studies have failed to detect significant HER?, homodimerization in solution or in crystals. The residues of domain II of EGFR and HER2 are similar. However, Arg285 at the dimer interface is not conserved between EGFR and HER2. In HER2 residue 2.85 is Leu.
  • HER2 is the only member of the ErhB family that does not bind known !igands. HER2 is instead activated via formation of heteromeric complexes with other ErbB family members and thereby indirectly regulated by EFGR and HERS ligands. HER2 is the preferred
  • HER2 enhances the affinity of the other ErbB receptors for their ligands by slowing down the rate of iigand-receptor complex dissociation, whereby HER2 enhances and prolongs signaling .
  • the ability of HER2 to enhance the iigand affinity of other ErbB receptors may reflect the promiscuous behavior of HER2 as a heterodimerization partner.
  • Heterodimerization of HER2 and another ligand-bound receptor of the ErbB family induces cross-phosphoryiation, leading to phosphorylation of the C-terminai tyrosine residues.
  • the most active HER2 heterodimer is the HER2-HER3 complex.
  • HER2 complements the kinase-deficient HER3 by providing an active kinase.
  • HER2 In contrast to EGFR, HER2 is internalization resistant when overexpressed. Overexpression of HER2 has further been reported to inhibit endocytosis of the other ErbB family members. Two mechanisms by which HER2 escapes lysosomal degradation and thereby remains at the plasma membrane have been suggested . Either HER2 avoids internalization or it becomes efficiently recycled from endosomes back to the plasma membrane. Studies using labeled antibodies have shown that HER2 is constantly internalized and recycled. Other studies in contrast failed to identify intracellular HER2 in cells treated with compounds known to inhibit recycling .
  • the third member of the ErbB family known as human epidermal growth factor receptor 3
  • HERS gene encodes a protein of 1342 amino acids with striking structural similarities to EGFR and HER2.
  • Features such as overall size, four extracellular subdomains (i-iV) with two cysteine clusters (domains II and IV), and a tyrosine kinase domain show structural similarities to EGFR and HER2.
  • the tyrosine kinase domain of HER3 shows 59% sequence homology to the tyrosine kinase domain of EGFR.
  • HER3 exists in a tethered conformation and an extended conformation, in the tethered conformation the dimerization arm is buried by interactions with domain IV, leaving domains I and HI too far apart for efficient ligand binding. Ligand binding to the extracellular domains I and HI occurs in the extended conformation of HER3 and leads to
  • HERS heterodimerization with other members of the ErbB family. No HERS homodimers are formed upon ligand binding.
  • the extended and ligand-bound HERS molecule preferentially
  • the tyrosine kinase of HER3 has impaired catalytic activity, insufficient for any detectable biological response.
  • Two amino acid residues which are highly conserved in the catalytic domains of protein kinases are altered in the catalytic domain of HER3. These are the substitution of aspargine for aspartic acid at residue 815 and substitution of histadine for glutamate at residue 740. The two amino acid substitutions may be the reason why HERS lacks catalytic activity of its tyrosine kinase domain. Because of the impaired intrinsic kinase activity of HER3 the receptor needs to heterodimerize with another ErbB family member in order to respond to its own ligand binding.
  • TKIs tyrosine kinase inhibitors
  • mAbs monoclonal antibodies
  • !igand-traps An advantage of monoclonal antibodies for treatment of cancer is target specificity, ensuring a low toxicity compared to conventional cytotoxic cancer chemotherapy.
  • Monoclonal antibodies have been approved for the treatment of solid tumors with abnormally high levels of EGFR or HER2, and numerous mAbs targeting EGFR or HER2 are in clinical trials.
  • TKIs inhibit receptor signaling by binding to the ATP-bindirtg site in the tyrosine kinase domain of EGFR and HER2.
  • Eriotinib/Tarceva® inhibits tyrosine kinases of EGFR while lapatinib/Tykerb® inhibits tyrosine kinases of both EGFR and HER2.
  • Both eriotinib and iaptinib are FDA approved TKis for use in the treatment of non-small iung cancer (NSCLC) and HER2 overexpressing metastatic breast cancer, respectively.
  • compositions targeting multiple EGFR-family members can be used to treat cancer, such as pancreatic, bone, colon, endometrial, or urinary tract cancer, including cancer that has acquired resistance to drug therapies targeting only one EGFR-family member.
  • the present invention is directed to humanized antibodies directed against EGFR, HER2 and HER3, as well as compositions comprising two or more humanized antibodies directed against two or more of these targets.
  • the invention is further directed to the use of the antibodies and compositions for human cancer therapy.
  • One aspect of the invention relates to a recombinant antibody composition
  • a recombinant antibody composition comprising at least one humanized anti-EGFR antibody or an antigen-binding fragment thereof, at least one humanized anti-HER2 antibody or an antigen-binding fragment thereof, and at least one humanized anti-HER3 antibody or an antigen-binding fragment thereof.
  • a humanized anti-EGFR antibody of the invention may be selected from an antibody comprising the heavy chain variable region sequence of SEQ ID NO: l and the light chain variable region sequence of SEQ ID NO: 3 or SEQ ID NO: 2, and an antibody comprising the heavy chain variable region sequence of SEQ ID NO:4 and the light chain variable region sequence of SEQ ID NO: 5.
  • the anti-EGFR antibody may comprise a heavy chain variable region sequence (SEQ ID NO: l) comprising Arg44 and Val83., and a light chain variable region sequence (SEQ ID NO: 2) comprising Ala i9 and Phe92; a heavy chain variable region sequence (SEQ ID NO : l) comprising Arg44, Vai83 and Ilel04, and a light chain variable region sequence (SEQ ID NO: 3) comprising Tyr41, Leu 51 and Phe92; or a heavy chain variable region sequence (SEQ ID NO: l) comprising Arg44, Val83 and Iiel04, and a iight chain variable region sequence (SEQ ID NO: 3) comprising Leu34, Tyr41, Leu51 and Phe92.
  • SEQ ID NO: 2 a heavy chain variable region sequence comprising Arg44 and Val83.
  • the anti-EGFR antibody may comprise a heavy chain variable region sequence (SEQ ID NO:4) comprising Leu20, Iie48 and Ala68, and a Iight chain variable region sequence (SEQ ID NO : 5) comprising Val75 and Phe87; or a heavy chain variable region sequence (SEQ ID NO :4) comprising Leu20, I!e48, Leu56, and Aia68, and a light chain variable region sequence (SEQ ID NO: 5) comprising Val75 and Phe87.
  • SEQ ID NO:4 comprising Leu20, Iie48 and Ala68
  • a Iight chain variable region sequence SEQ ID NO : 5
  • SEQ ID NO:4 comprising Leu20, I!e48, Leu56, and Aia68
  • the invention encompasses a humanized anti -EGFR antibody whose heavy and light chain amino acid sequences comprise : SEQ ID Os:43 and 44, respectively, SEQ ID NOs : 38 and 39, respectively, SEQ ID NOs :41 and 42, respectively, SEQ ID NOs :45 and 46, respectively, or SEQ ID NQs:47 and 48, respectively, or an antigen-binding fragment thereof.
  • a humanized anti-HER2 antibody of the invention may be selected from an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NQ: 7, and an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 8 and the Iight chain variable region sequence of SEQ ID NO: 9.
  • the anti-HER2 antibody may comprise a heavy chain variable region sequence (SEQ ID NO: 6) comprising Ser55, Leu70, Va!72, Lys74 and Ala79, and a light chain variable region sequence (SEQ ID NO: 7) comprising Va!44, Met48 and Tyr70; or a heavy chain variable region sequence (SEQ ID NO: 6) comprising Ser55 and Val72, and a light chain variable region sequence (SEQ ID NO: 7) comprising Met48 and Tyr70.
  • SEQ ID NO: 6 heavy chain variable region sequence
  • SEQ ID NO: 7 comprising Met48 and Tyr70.
  • the anti-HER2 antibody may comprise a heavy chain variable region sequence (SEQ ID NO: 8) comprising Ala49, ile74 and Ser77, and a Iight chain variable region sequence (SEQ ID NO: 9) comprising Thr56, Tyr71, Ser85 and Leu 104.
  • the invention encompasses a humanized anti-HER2 antibody whose heavy and light chain amino acid sequences comprise : SEQ ID NOs:51 and 52, respectively, SEQ ID NOs :49 and 50, respectively, or SEQ ID NOs : 53 and 54, respectively, or an antigen-binding fragment thereof.
  • a humanized anti-HER3 antibody of the invention may be selected from an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 10 and the Iight chain variable region sequence of SEQ ID NO: 11, and an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 12 and the light chain variable region sequence of SEQ ID NO : 13.
  • the anti-H ER3 antibody may comprise a heavy chain variable region sequence (SEQ ID NO : 10) comprising et49, Ser55 and I!e68, or Asn44, Ser55 and Thr93, and a light chain variable region sequence (SEQ ID NO : 1.1) comprising Phe36, Val44, Phe49 and Iie85, or Phe36, Phe49 and Leu73.
  • the anti-HER3 antibody may comprise a heavy chain variable region sequence (SEQ ID NO : 1.2) comprising Val46, Met49, Ser55 and Arg72, and a light chain variable region sequence (SEQ ID NO : 13) comprising Val21, Vai44 and Phe87, and optionally Thr29; or a heavy chain variable region sequence (SEQ ID NO : 12) comprising Phe41 , Val46, Met49, Ser55 and Arg72 f and a light chain variable region sequence (SEQ ID NO : 13) comprising Val21 , Vai44, Tyr71, Phe87 and Leu 104,
  • the i nvention encompasses a hu manized anti-HER3 a ntibody whose heavy and lig ht chain a mi no acid sequences com prise : SEQ ID NOs : 55 a nd 56, respectively, SEQ ID NOs : 57 a nd 58, respectively, SEQ ID NOs : 59 a nd 60, respectively, or SEQ ID NOs : 61 a nd 62, respectively, or an a ntigen-bi nding frag ment thereof.
  • the invention also encompasses antibody compositions comprising two, three, four, five or six of the antibodies described above.
  • the antibody composition may comprise (i) 11294 and/or 11302; (ii) 11249 and/or 11145 ; and (iii) 10738 and/or 11052.
  • the composition comprises all six antibodies.
  • the antibody composition may comprise (a) anti-EGFR antibody 1.0292, 10460, or 11294; (b) anti-EGFR antibody 10560 or 11302 ; (c) anti-H ER2 antibody 10704 or 11249; (d) anti-H ER2 antibody 111.45 ; (e) anti-H ER3 antibody 10738 or 1.0810; and (f) anti-H ER3 antibody 11006 or 11052.
  • the antibody composition comprises anti-EGFR antibodies 11294 and 1.1302, anti-HER2 antibodies 112.49 and 111.45, and anti-H ER3 antibodies 10738 and 11052.
  • Antibody 10292, 1.0460, 11294, 10560, 11.302, 10704, 11249, 1 1145, 10738, 1.0810, 11006, or 11052 may comprise at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4.
  • the antibody composition may comprise (a) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 43 and the light chain variable region sequence of SEQ ID O : 44; (b) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 47 and the light chain variable region sequence of SEQ ID NO : 48; (c) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 51 and the light chain variable region sequence of SEQ ID NO : 52; (d) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 53 and the light chain variable region sequence of SEQ ID NO : 54; (e) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 55 and the light chain variable region sequence of SEQ ID NO : 56; and (f) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 61 and the light chain variable region sequence of SEQ ID NO: 62.
  • Further aspects of the invention relate to a method for producing antibodies and antibody compositions of the invention; a pharmaceutical composition comprising an antibody or an antibody composition of the invention and a pharmaceutically acceptable diluent, carrier, or exdpient; a method for treating cancer in a human or other mammal comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant antibody composition or pharmaceutical composition of the invention; use of a recombinant antibody composition or a pharmaceutical composition of the invention for preparing a medicament for the treatment of cancer; and a recombinant antibody composition or pharmaceutical of the invention for use as a medicament for treatment of cancer.
  • the antibodies preferably are directed to human HER family members, in some embodiments, each of these compositions comprises more than one monoclonal antibody, each binding to a different epitope in the targeted HER.
  • at least one of the antibodies is conjugated to an anti-cancer agent, e.g., a cytotoxic agent, a cytokine, a toxin, or a radionuclide.
  • Cancer treatable by the methods of the invention includes, without limitation, pancreatic cancer (including pancreatic cancer facilitated by a KRAS mutation), head and neck cancer, breast cancer, bone cancer, colon (including colorectal cancer) cancer, endometrial cancer, urinary tract cancer, skin cancer, lung cancer, prostate cancer, gastric cancer, esophageal cancer, ovarian cancer, other epidermal cancer, and cancers with a dependency on one or more of EGFR, HER2, and HER3.
  • pancreatic cancer including pancreatic cancer facilitated by a KRAS mutation
  • head and neck cancer breast cancer
  • bone cancer including colorectal cancer
  • colon cancer including colorectal cancer
  • endometrial cancer urinary tract cancer
  • skin cancer skin cancer
  • lung cancer prostate cancer
  • gastric cancer gastric cancer
  • esophageal cancer ovarian cancer
  • other epidermal cancer other epidermal cancer
  • the patient may have been treated for cancer previously.
  • the patient may have been treated with a drug targeting a single EGFR-family member and have acquired resistance to the drug (e.g., cetuximab, trastuzumab, or pertuzumab).
  • a drug targeting a single EGFR-family member e.g., cetuximab, trastuzumab, or pertuzumab.
  • the invention also relates to a nucleic acid molecule comprising a nucleotide sequence encoding any of the antibody heavy or light chains or heavy or light variable regions described herein.
  • the invention also relates to an expression vector comprising such nucleic acid molecules and a host cell comprising such nucleic acid molecules or vectors.
  • the host cell may be capable of expressing any of the antibodies described herein.
  • Figure 1 Amino acid sequence alignment of variable chains of the anti-EGFR humanized monoclonal antibodies 10292, 10460, and 11294 with the in siii ' co designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT.
  • Top Variable heavy chains of 10292 (SEQ ID NO: 38), 10460 (SEQ ID NO:40), and 112.94 (SEQ ID NQ:42) aligned to CDR grafted seguence (1277_ CDRgrafted-H ; SEQ ID f ⁇ iO: 62) .
  • Middle Variable light chain of 10292. (SEQ ID NO : 39) aligned to CDR grafted sequence (1277_CDRgrafted-L; SEQ ID NQ: 63) .
  • Bottom Variable light chains of 1.0460 (SEQ ID NO:41 ) and 112.94 (SEQ ID NO:43) aligned to CDR grafted sequence (1277A_CDRgrafted-L; SEQ ID NQ: 64) .
  • Figure 2 Amino acid sequence alignment of variable chains of the anti-EGFR humanized monoclonal antibodies 10560 and 11302 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT.
  • Top Variable heavy chains of 10560 (SEQ ID NO:44) and 11302 (SEQ ID NO :46) aligned to CDR grafted sequence (1565 .. CDRgrafted-H ; SEQ ID NO: 65) .
  • Figure 3 Amino acid sequence alignment of variable chains of the anti-HER2 humanized monoclonal antibodies 10704 and 11249 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT.
  • Top Variable heavy chains of 10704 (SEQ ID NO:48) and 11249 (SEQ ID NO : 50) aligned to CDR grafted sequence (4384_CDRgrafted-H ; SEQ ID NO: 67) .
  • Bottom Variable light chains of 10704 (SEQ ID NO:49) and 11249 (SEQ ID NO: 51) aligned to CDR grafted sequence (4384_CDRgrafted-L; SEQ ID NO: 68) .
  • Figure 4 Amino acid sequence alignment of variable chains of the anti-HER2 humanized monoclonal antibody 11145 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT.
  • Top Variable heavy chain of 11145 (SEQ ID NO: 52) aligned to CDR grafted sequence (4517_CDRgrafted-H ; SEQ ID NO: 69) .
  • Bottom Variable light chain of 11 145 (SEQ ID NO: 53) aligned to CDR grafted sequence (4517_CDRgrafted-L; SEQ ID NO: 70) .
  • Figure 5 Amino acid sequence alignment of variable chains of the anti-HER3 humanized monoclonal antibodies 10738 and 10810 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT.
  • Top Variable heavy chains of 10738 (SEQ ID NO: 54) and 10810 (SEQ ID NO : 56) aligned to CDR grafted sequence (5G38 . du . CDRgrafted-H ; SEQ ID NO: 71).
  • Bottom Variable light chains of 10738 (SEQ ID NO: 55) and 10810 (SEQ ID NO: 57) aligned to CDR grafted sequence (5038_CDRgrafted-L; SEQ ID NO: 72).
  • Figure 6 Amino acid sequence alignment of variable chains of the anti-HER3 humanized monoclonal antibodies 11006 and 11052 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT.
  • Top Variable heavy chains of 11006 (SEQ ID NO: 58) and 11052 (SEQ ID NO: 60) aiigned to CDR grafted sequence (5082_CDRgrafted-H; SEQ ID NO: 73).
  • Figure 7 In vitro activity of humanized anti-EGFR antibody variant 10292 in combination with its chimeric anti-EGFR partner antibody.
  • A431NS cells (top panel) and H358 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 8 In vitro activity of humanized anti-EGFR antibody variant 10460 in combination with its chimeric anti-EGFR partner antibody.
  • A431NS cells (top panel) and H358 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 9 In vitro activity of humanized anti-EGFR antibody variant 10560 in combination with its chimeric anti-EGFR partner antibody.
  • A431NS cells (top panel) and H358 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 10 In vitro activity of humanized anti-HER2 antibody variant 10704 in combination with its chimeric anti-HER2 partner antibody. OE19 ceils (top panel) and BT474 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 11 In vitro activity of humanized anti-HER2 antibody variant 11145 in combination with its chimeric anti-HER2 partner antibody. OE19 ceils (top panel) and BT474 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 12 In vitro activity of humanized anti-HER3 antibody variant 10738 in combination with its chimeric anti-HER3 partner antibody.
  • MBA-MD-175 VII cells top panel
  • MCF-7 cells in the presence of 1 n heregulin beta; bottom panel
  • Data are presented as means ⁇ SEM.
  • Figure 13 In vitro activity of humanized anti-HER3 antibody variant 10810 in combination with its chimeric anti-HER3 partner antibody.
  • MBA-MD-175 VII ceils top panei
  • MCF-7 celis in the presence of 1 n heregulin beta; bottom panei
  • Data are presented as means ⁇ SEM.
  • Figure 14 In vitro activity of humanized anti-HER3 antibody variant 11006 in combination with its chimeric anti-HER3 partner antibody.
  • MB.A-MD-175 VII ceils top panei
  • MCF-7 cells in the presence of 1 nM heregulin beta; bottom panei
  • Data are presented as means ⁇ SEM.
  • Figure 15 In vitro activity of humanized anti-HER3 antibody variant 11052 in combination with its chimeric anti-HER3 partner antibody.
  • MBA-MD-175 VII ceils top panei
  • MCF-7 celis in the presence of 1 nM heregulin beta; bottom panei
  • Data are presented as means ⁇ SEM.
  • Figure 16 Cross-reactivit pattern of chimeric and humanized antibodies with human, cynomolgus and murine HER family antigens.
  • Figure 17 In vitro activity of humanized anti-EGFR antibody variant 11294 in combination with its chimeric anti-EGFR partner antibody.
  • A431NS cells (top panel) and FaDu ceils (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 18 In vitro activity of humanized anti-EGFR antibody variant 11302 in combination with its chimeric anti-EGFR partner antibody. A431 NS ceils (top panel) and FaDu ceils (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • FIG 19 In vitro activity of humanized anti-HER2 antibody variant 11249 in combination with its humanized anti-HER2 partner antibody 11145. OE19 ceils (top panei) and BT474 ceils (bottom pane!) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 20 In vitro activity of a mixture of humanized antibodies (variants 11294, 11302, 11249, 11145, 10738 and 11052; humanized Pan-HER) and a mixture of chimeric antibodies (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER).
  • the indicated ceil lines were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ⁇ SEM.
  • Figure 21A is a schematic illustrating the interaction of Pan-HER with its EGFR (left), HER2 (middle) and HER3 (right) target proteins.
  • Figure 21B Is a series of charts showing the effects of treatment with EGFR (left), HER2 (middle) and HER3 (right) antibodies on the metabolic activity of A431N5, HCC202, and MDA- MB-175-VII ceil lines, respectively.
  • the figure legend in the left panel lists from top to bottom: Negative controi, 1277, 1565, 12774- 1565.
  • the figure legend in the center panel lists from top to bottom : Negative control, 4384, 4517, 43844-4517.
  • the figure legend in the right panel lists from top to bottom : Negative control, 5038, 5082, 5038+5082.
  • Figure 21C is a series of Western blot images showing the levels EGFR (left), HER2 (middle), and HER3 (right) in the total cell lysates of A431NS, HCC202 and MDA-MB-175-VII cancer cells, respectively, that had been treated with the indicated antibodies and antibody mixtures.
  • Figure 22 is an image showing the receptor phosphorylation levels of EGFR (left), HER2 (middle), and HER3 (right) in 73 cancer cell lines treated with Pan -HER (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER).
  • Figure 23 is a table showing maximal metabolic activity as a percentage of untreated (no Heregulin or EGF) control cells (set to 100%) after treatment with Pan-HER mixture (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER), Pan-HER subcomponents and a negative controi antibody.
  • EGF Heregulin
  • Figure 24 is a table showing maximal metabolic activity as a percentage of untreated control cells in the absence of ligand (set to 100%) after treatment with Pan-HER (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER), Pan-HER subcomponents and a negative controi antibody in the presence of 5 nM Heregulin.
  • Cells were exposed to medium containing antibodies and ligands for 96 hours, (i.e. ligand and antibody was added simultaneously to the cells).
  • Figure 25 is a table showing maximal metabolic activity as a percentage of untreated control cells in the absence of ligand (set to 100%) after treatment with Pan-HER (12.77, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER), Pan-HER subcomponents and a negative control antibody in the presence of 1 nM EGF. Ceils were exposed to medium containing antibodies and ligands for 96 hours, (i.e. iigand and antibody was added simultaneously to the ceils).
  • Figure 26 is an image showing the mutation status of genes listed across the top of the image of seven pancreatic cancer ceil lines (CAPAN-1, PK-1, CFPAC-1, BxPC3 ; ASPC1, CAPAN-2, Pan08.13 f PANC-1, KP4, iaPaca-2 and PSN1).
  • Figure 27 is a series of graphs showing the dose-response of the CAPAN-1 cell line to Pan-HER treatment in the absence (left) or presence of Heregulin (middle) and EGF (right) ligands.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • FIG. 25 is a series of graphs showing the effects of Pan-HER and reference antibodies on the metabolic activity of parental cell lines (top) and the corresponding resistant clones that have acquired resistance to cetuximab, trastuzumab or pertuzumab (bottom).
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • the figure legend in the left top panel lists from top to bottom : Pan- HER, cetuximab, Neg. control.
  • the figure legend in the center top panel lists from top to bottom : Pan-HER, trastuzumab, Neg. control.
  • the figure legend in the right top panel lists from top to bottom: Pan-HER, pertuzumab, Meg. control.
  • Figure 28 is a series of graphs showing the effects of Pan-HER and reference antibodies on the metabolic activity of parental cell lines (top) and the corresponding resistant clones that have acquired resistance to cetuximab, trastuzumab or pertuzumab (bottom).
  • Pan -HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • the figure legend in the left top panel lists from top to bottom : Pan- HER, cetuximab, Neg. control.
  • the figure legend in the center top panel lists from top to bottom : Pan-HER, trastuzumab, Neg. control.
  • the figure legend in the right top panel lists from top to bottom: Pan-HER, pertuzumab, Neg. control.
  • Figure 29 is a series of Western blot images showing the levels of EGFR, HER2 and HER3 in whole ceil lysates of H292 (top) and OVCAR-8 (bottom) cell lines after antibody treatment.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • Figure 30 is a graph showing the effects of treatment with Pan-HER or its subcomponents on tumor volume in the BxPC-3 xenograft model.
  • Pan-HER refers to a mixture of antibodies
  • EGFR refers to a mixture of antibodies 1277 and
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR+HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+ HER3 refers to a mixture of antibodies 1277., 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Figure 31 is a series of images showing EGFR and HER2 immunoiabeled sections of tumors resected from vehicle and Pan-HER treated BxPC-3 xenografts three days after withdrawal of treatment.
  • Pan-HER refers to a mixture of antibodies 12.77, 1565, 4384, 4517, 5038, and 5082.
  • Figure 32 is a graph showing the effects of treatment with Pan-HER or its subcomponents on tumor volume in the Calu-3 xenograft model .
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HERS refers to a mixture of antibodies 5038 and 5082.
  • EGFR+ HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+ HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Figure 33 is a series of Western blot images showing the levels of EGFR, HER2, HERB and a ⁇ -actin loading control in BxPC-3 tumor iysates after antibody treatment.
  • the relative quantification of EGFR, HER2, and HERB levels in the Western blot band intensities is shown in a series of charts in Fig . 30 (bottom) .
  • “Pan-HER” refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR+ HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+ HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+ HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Figure 34 is a series of graphs showing the effects of Pan-HER on tumor volume in ST191, ST204, ST383, STS021, ST179, ST385, STS064, ST334, STS059, and STS058 patient-derived tumor xenograft models of KRAS mutated pancreatic cancer.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • Figure 35 is a series of graphs showing the effects of treatment with Pan-HER or its subcomponents on tumor volume in ST179 and ST383 patient-derived tumor xenograft models of KRAS mutated pancreatic cancer.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR+ HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+ HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2 -HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Figure 36 is a schematic illustrating the development and cloning of acquired cetuximab resistant HNS clones.
  • Figure 37 is a graph showing the dose-response effects of cetuximab treatment on parental HNS ceils and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14.
  • Figure 38 is a graph showing the binding curve of cetuximab to fixed parental HNS cells and cetuximab resistant clones HN5 CR2, HNS CR6, HNS CR13, and HNS CR14.
  • Figure 39 is a graph showing the relative surface levels of EGFR found by fluorescence flow cytometry in parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HN5 CR13, and HNS CR14,
  • Figure 40 is a series of Western blot images showing the total levels of EGFR, phosphorylated EGFR species, and a ⁇ -actin loading control in cell lysates from parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 that were either untreated (left) or stimulated with EGF (right).
  • Figure 41 is a series of Western blot images showing the total levels of EGFR, AKT, pAKT (Ser473), ERK1/2, pERKl/2(Thr202/Tyr204), and a ⁇ -actin loading control in cell lysates from parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 that were either untreated (left) or stimulated with EGF (right).
  • Figure 42 is a graph showing the viability of parental HNS cells and cetuximab resistant clones HNS CR2 and HNS CR14 treated with EGFR-LNA, cetuximab, EGFR-2mix (antibodies 1277 and 1565) or controls.
  • Figure 43 is a series of Western blot images showing the total levels of EGFR in parental HNS cells and cetuximab resistant clones HNS CR2 and HN5 CR14 treated with EGFR-LNA, cetuximab, EGFR-2mix (antibodies 1277 and 1.565) or controls.
  • Figure 44 is a graph showing the viability of parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 treated with the indicated EGFR antibodies.
  • EGFR 2mix refers to a mixture of antibodies 1277 and 1565.
  • Her3 2mix refers to a mixture of antibodies 5038 and 5082.
  • Figure 45 is a series of graphs showing the dose-response of parental HNS ceils (Fig. 45A) and cetuximab resistant clones HNS CR2 (Fig. 45B) viability to treatment with the indicated antibodies.
  • EGFR 2mix refers to a mixture of antibodies 1277 and 1565.
  • Her3 2mix refers to a mixture of antibodies 5038 and 5082.
  • EGFR+HER3 4mix refers to a mixture of antibodies 1277., 1565, 5038 and 5082.
  • antibody mixtures targeting EGFR-famiiy members EGFR, HER2, and HER3 effectively suppress tumor growth in multiple xenograft models of human cancer, including hard-to-treat patient-derived models of pancreatic cancer.
  • antibody mixtures targeting more than one EGFR-famiiy member retain their inhibitory effect in cells that have acquired resistance to therapeutic monoclonal antibodies such as cetuximab, trastuzumab, and pertuzumab.
  • One aspect of the invention relates to humanized antibodies that bind the EGFR-famiiy members EGFR, HER2, and HER3.
  • the term "antibody” or " antibody molecule” describes a functional component of serum and is often referred to either as a collection of molecules
  • antibodies or immunoglobulin or as one molecule (the antibody molecule or immunoglobulin molecule).
  • An antibody is capable of binding to or reacting with a specific antigenic
  • an individual antibody is usually regarded as
  • a composition of antibodies may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of two or more different antibodies reacting with the same or different epitopes on the same antigen or even on distinct, different antigens).
  • Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, and ail natural antibodies have the same overall basic structure of two identical light chains and two identical heavy chains.
  • Antibodies are also known collectively as immunoglobulins.
  • antibody or “antibodies” as used herein are intended to include single chain antibodies as well as binding fragments of antibodies, such as Fab, F(ab')2, Fv fragments or single chain Fv (scFv) fragments, and multimeric forms such as dimeric igA molecules or pentavalent IgM.
  • references to an “antibody” or “antibodies” are therefore intended to encompass, in particular, binding fragments and single chain antibodies, unless it is indicated otherwise or apparent from the context that this is not the case.
  • Each heavy chain of an antibody typically includes a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region typically includes three domains, referred to as CHI, CH2 and CHS.
  • Each antibody light chain typically includes a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region typically includes a single domain, referred to as CL.
  • the VH and VL regions may be further subdivided into regions of hypervariability ("hypervariable regions", which may be
  • variable domains of an antibody that are primarily responsible for determining the antibody's binding specificity. These are also referred to as complementarity determining regions (CDRs), which are interspersed with regions that are more conserved, termed framework regions (FRs) .
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each of the heavy and light chains of an antibody contains three CDR regions, referred to as CDRi, CDR2 and CDRS, of which CDRS snows the greatest variability.
  • CDRi, CDR2 and CDRS regions that are more conserved, termed framework regions (FRs) .
  • FRs framework regions
  • Each of the heavy and light chains of an antibody contains three CDR regions, referred to as CDRi, CDR2 and CDRS, of which CDRS snows the greatest variability.
  • Each VH and VL typically includes three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FRi, CDRI, FR2, CDR2,
  • amino acid residues in the variable regions are often numbered using a standardized numbering method known as the Kabat numbering scheme (Kabat et ai. (1991) Sequences of Proteins of Immunological interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, USA), although other numbering schemes such as Chothia and IMGT also exist.
  • recombinant antibody refers to an antibody that is expressed from a ceil or ceil line transfected with an expression vector (or possibly more than one expression vector, e.g. two expression vectors) comprising the coding sequence of the antibody, where said coding sequence is not naturally associated with the cell.
  • Table 1 below shows the SEQ ID NOs, as set forth in Table 8, for the DNA and amino acid sequences of the heavy chain variable regions (VH) and the light chains (LC) of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • TabUe 1 SEQ ID NOs for the DMA and amino acid sequences of the heavy chairs variable re io s and li ht chains of chimeric antibodies
  • the specificity of an antibody's interaction with a target antigen resides primarily in the amino acid residues located in the six CDRs of the heavy and light chain.
  • the amino acid sequences within CDRs are therefore much more variable between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally any specific antibody with a given amino acid sequence, by constructing expression vectors that express CDR sequences from the specific antibody grafted into framework sequences from a different antibody. As a result, it is possible to "humanize" a non- human antibody and still substantially maintain the binding specificity and affinity of the original antibody. A more detailed discussion of humanization is provided below.
  • a “chimeric antibody” refers in its broadest sense to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically an antibody that is partially of human origin and partially of non-human origin, i .e. derived in part from a non-human animal, for example a mouse, rat or other rodent, or an avian such as a chicken. Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of a human anti-antibody response, e.g . a human anti-mouse antibody response in the case of a murine antibody.
  • variable region sequences are murine sequences derived from immunization of a mouse, while the constant region sequences are human.
  • non-human parts may be subjected to further alteration in order to humanize the antibody.
  • present invention is based on humanization of certain chimeric antibodies having murine variable region sequences.
  • humanize refers to the fact that where an antibody is wholly or partially of non- human origin, for example a murine antibody obtained from immunization of mice with an antigen of interest or a chimeric antibody based on such a murine antibody, it is possible to replace certain amino acids, in particular in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize an immune response in humans, it is known that ail antibodies have the potential for eliciting a human anti-antibody response, which correlates to some extent with the degree of "humanness" of the antibody in question. Although it is not possible to precisely predict the immunogenicity and thereby the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic than human antibodies.
  • Chimeric antibodies where the foreign (usually rodent) constant regions have been replaced with sequences of human origin, have been shown to be generally less immunogenic than antibodies of fully foreign origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies.
  • chimeric antibodies or other antibodies of non-human origin it is therefore preferred that they be humanized to reduce the risk of a human anti-antibody response.
  • humanization typically involves modification of the framework regions of the variable region sequences.
  • Amino acid residues that are part of a complementarity determining regions (CDRs) will most often not be altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a giycosylation site, a deamidation site, an aspartate isomerization site or an undesired cysteine or methionine residue.
  • CDRs complementarity determining regions
  • Removal of an N-glycosyiation site may be achieved by mutating either the Asn or the Ser/Thr residue to a different residue, preferably by way of conservative substitution.
  • Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure.
  • Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Aia. When such a deamidation site, in particular Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues.
  • CDR grafting which for e.g. a murine-derived chimeric antibody involves identification of human germline gene counterparts to the murine variable region genes and grafting of the murine CDR sequences into this framework. CDR grafting may be based on the
  • CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR-grafted non-human antibody
  • back mutations may be introduced at selected positions of the CDR-grafted antibody, typically in the framework regions, in order to reestablish the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molecule, while residues that are buried or that have a low degree of surface exposure will not normally be altered .
  • An alternative humanlzation technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non- human origin are retained, while surface residues are altered to human residues.
  • affinity maturation it may also be desirable to alter one or more CDR amino acid residues in order to improve binding affinity to the target epitope.
  • This is known as "affinity maturation” and may optionally be performed in connection with humanlzation, for example in situations where humanization of an antibody leads to reduced binding specificity or affinity and it is not possible to sufficiently improve the binding specificity or affinity by back mutations alone.
  • affinity maturation methods are known in the art, for example the in vitro scanning saturation mutagenesis method described by Burks et al . (1997) PNAS USA, vol . 94, pp. 412- 417 and the stepwise in vitro affinity maturation method of Wu et al . (1998) PNAS USA, vol . 95, pp. 6037-6042.
  • Amino acid residues herein may be indicated by either the one-letter code or the three-letter code.
  • Amino acid substitutions relative to a reference sequence may e.g . be indicated using the format "G44R", which indicates that a glycine residue in position 44 of a reference sequence has been mutated to an arginine residue.
  • G44R indicates a mutation of the glycine residue in a CDR-grafted antibody to an arginine residue.
  • An amino acid residue written in the format "Arg44” indicates a particular residue in a particular position, i.e. in this case an arginine residue in position 44. Unless otherwise indicated, numbering of amino acid residues refers to the appended sequence listing.
  • the present invention relates to humanized antibodies, more particularly to humanized antibodies based on certain chimeric parent antibodies described in WO
  • humanized antibodies of the invention were developed using CDR grafting and back mutations, and in some cases alteration of unwanted sequence motifs, starting with selected chimeric anti-EGFR, anti-HER2 and anti-HER3 antibodies described in WO
  • antibody 10560 refers to the specific humanized antibodies described below, which have been prepared by CDR grafting based on a chimeric parent antibody.
  • antibody 10560 is an antibody with a heavy chain comprising the heavy chain variable region sequence (VH) set forth in SEQ ID NO:4 and a light chain comprising the light chain variable region sequence (VL) set forth in SEQ ID NO: 5, and comprising substitutions (for example, back mutations) at certain positions compared to the original CDR-grafted antibody (see Table 3 and Figures 1-6).
  • the antibodies also included a human kappa constant region sequence (SEQ ID NO:42 in WO 2012/059858 and US 2011/0217305, with an N-terminai Arg residue) and a human IGHG1 heavy chain constant region sequence (SEQ ID NO:44 in WO 2012/059858 and US
  • humanized antibodies of the invention are described herein by way of an antibody number, i.e. 10292, 10460, 11294, 10560, 10704, 11302, 11145, 11249, 10738, 10810, 11006 or 11052. These are derived from the chimeric antibodies (murine variabie regions, human constant regions) disclosed in WO 2012/059857 by CDR grafting and subsequent mutation at certain positions, primarily back mutations, as described in Example 1. Table 2 below outlines how the humanized antibodies of the invention are related to the chimeric parent antibodies disclosed in WO 2012/059857.
  • Table 3 below provides the SEQ ID NOs of exemplary humanized antibodies of the Invention, as well as the individual substitutions (back mutations, and in certain cases mutation(s) to alter undesired sequence motifs) in the heavy chain (HC) and light chain (LC) compared to the original CDR-grafted antibody.
  • the amino acid sequences of the heavy and light chains of the antibodies listed in Table 3 are provided in Figures 1-6 and in separate SEQ ID NOs enclosed in parentheses in Tabie 3.
  • the CDR sequences in Figures 1-6 are indicated with shading.
  • any of the numbered humanized antibodies listed in Table 2 may comprise "at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4" means that the antibodies may comprise additional substitutions in one or more "Xaa” residues other than the substitutions listed above in Table 3.
  • Xaa can be any na rally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurring amino acid
  • Xaa can be any naturally occurrincj amino acid
  • Gly Asn lie Asn Pro Ser Xaa Gl Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60
  • One aspect of the invention relates to particular humanized antibodies targeting EGFR., HER2. or HER3, These individual antibodies include the following :
  • the above-outlined humanized antibodies typically include, in both the heavy chain variable region sequence and the light chain variable region sequence, one or more of the possible substitutions (primarily back mutations, but in certain cases also mutation to alter unwanted sequence motifs) set forth in Table 4 and in the examples and accompanying figures.
  • the heavy chain variable region sequence and the light chain variable region sequence will typically each comprise two, three, four or five such substitutions.
  • Examples of a preferred anti-EGFR antibody (a) are antibodies comprising:
  • a heavy chain variable region sequence (SEQ ID NO: l) comprising Arg44 and Vai83, and a light chain variable region sequence (SEQ ID NO: 2) comprising Aial9 and Phe92. [e.g., antibody 10292] ; (ii) a heavy chain variabie region sequence (SEQ ID NO: i) comprising Arg44, Vai83 and Ilel04, and a light chain variabie region sequence (SEQ ID NO: 3) comprising Tyr41, Leu 51 and Phe92 [e.g.., antibody 10460] ; or
  • a heavy chain variabie region sequence (SEQ ID NO: 1) comprising Arg44, Vai83 and Iiel04, and a iight chain variabie region sequence (SEQ ID NO: 3) comprising Leu34, Tyr41, Leu 51 and Phe92 [e.g., antibody 11294] .
  • the anti-EGFR antibody (a) may also be an antibody corresponding to antibody 10292, 10460, or 11294, but comprising at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4, e.g. substitution in one, two, three or four of such "Xaa” residues.
  • SEQ ID NO: 2 includes Xaa in positions 33-34, since the CDR-grafted sequence has a deamidation site (Asn-Gly) in these positions. Although it is possible to perform substitutions in both positions, it is sufficient to alter only one of the two positions in order to eliminate the deamidation site. The sequence will therefore typically include either Asn in position 33 or Gly in position 34.
  • An example of a preferred anti-EGFR antibody (b) is one comprising:
  • a heavy chain variable region sequence (SEQ ID NO:4) comprising Leu20, I!e48 and Aia68, and a light chain variable region sequence (SEQ ID NO: 5) comprising Val75 and Phe87 [e.g., antibody 10560] ; or
  • a heavy chain variable region sequence (SEQ ID NO:4) comprising Leu20, I!e48, Leu56, and Ala68, and a iight chain variabie region sequence (SEQ ID NO: 5) comprising Val75 and Phe87 [e.g., antibody 11302] .
  • the anti -EGFR antibody (b) may also be an antibody corresponding to antibody 10560 or 11302, but comprising at least one additional substitution in any of the heavy chain and/or Iight chain amino acid residues indicated as "Xaa” in Table 4, e.g. substitution in one, two, three or four of such "Xaa” residues.
  • SEQ ID NO:4 includes Xaa in positions 55-56, since the CDR-grafted sequence has a deamidation site (Asn-Gly) in these positions. Although it is possible to perform substitutions in both positions, it is sufficient to alter only one of the positions in order to eliminate the deamidation site. The sequence will therefore typically include either Asn in position 55 or Gly in position 56.
  • An example of a preferred anti-HER2 antibody (c) is one comprising:
  • a heavy chain variable region sequence (SEQ ID NO: 6) comprising Ser55, Leu70, Vai72, Lys74 and Ala79, and a iight chain variabie region sequence (SEQ ID NO: 7) comprising Vai44, Met48 and Tyr70 [e.g., antibody 10704] ; or
  • the anti-HER2 antibody (c) may also be an antibody corresponding to antibody 10704 or 1.1249, but comprising at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4, e.g. substitution in one, two, three or four of such "Xaa” residues.
  • An example of a preferred anti-HER2 antibody (d) is one comprising a heavy chain variable region sequence (SEQ ID NO:8) comprising Aia49, Iie74 and Ser77, and a light chain variable region sequence (SEQ ID NO:9) comprising Thr56, Tyr71, Ser85 and Leul04 [e.g., antibody 11145] .
  • the anti-HER2 antibody (d) may also be an antibody corresponding to antibody 11145, but comprising at ieast one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4, e.g. substitution in one, two, three or four of such "Xaa” residues.
  • Examples of a preferred anti-HER3 antibody are antibodies comprising a heavy chain variable region sequence (SEQ ID NO: 10) comprising et49, Ser55 and Iie68, or comprising Asn44, Ser55 and Thr93, and a light chain variable region sequence (SEQ ID NO: 11) comprising Phe36, Val44, Phe49 and Iie85, or comprising Phe36, Phe49 and Leu73.
  • a heavy chain variable region sequence SEQ ID NO: 10
  • Ser55 and Iie68 or comprising Asn44, Ser55 and Thr93
  • a light chain variable region sequence SEQ ID NO: 11
  • Particular examples of such anti-HER3 antibodies are those comprising :
  • a heavy chain variable region sequence (SEQ ID NO: 10) comprising Met49, Ser55 and ile68, and a light chain variable region sequence (SEQ ID NO: 11) comprising Phe36, Va!44, Phe49 and Ile85 [e.g., antibody 10738] ; or
  • a heavy chain variable region sequence (SEQ ID NO: 10) comprising Asn44, Ser55 and Thr93, and a light chain variable region sequence (SEQ ID NO: 11) comprising Phe36, Phe49 and Leu73 [e.g., antibody 10810] .
  • the anti-HER3 antibody (e) may also be an antibody corresponding to antibody 10738 or 10810, but comprising at Ieast one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4, e.g. substitution in one, two, three or four of such "Xaa” residues.
  • Examples of a preferred anti-HER3 antibody (f) are antibodies comprising :
  • a heavy chain variable region sequence (SEQ ID NO: 12) comprising Va!46, Met49, Ser55 and Arg72, and a light chain variable region sequence (SEQ ID NO: 13) comprising Vai21, Val44 and Phe87, and optionally Thr29 [e.g., antibody 11006] ; or
  • the anti-HER3 antibody (f) may also be an antibody corresponding to antibody 11006 or 1.1052, but comprising at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa” in Table 4, e.g. substitution in one, two, three or four of such "Xaa” residues.
  • antibodies exist as different isotypes, such as the human isotypes IgGl, IgG2, IgG3, IgG4, IgAl and igA2, or the murine isotypes IgGl, igG2a, igG2b, IgG3 and IgA,
  • An antibody of the invention may be of any isotype, including IgG, IgM, IgE, IgA, or igD.
  • a further aspect of the invention relates to a recombinant antibody composition (or mixture) comprising at least two humanized antibodies of the invention directed against at least two different receptors selected from EGFR, HER2 and HER3.
  • polyclonal antibody or “mixture of [monoclonal] antibodies” refer to a composition of two or more different antibody molecules which are capable of binding to or reacting with different specific antigenic determinants on the same or on different antigens, in the context of the present invention, the individual antibodies of a mixture of antibodies bind to different antigenic determinants of at least two HER family receptors.
  • the individual antibodies preferably bind to different epitopes of that receptor, more preferably distinct and substantially non-overlapping epitopes.
  • pan- HER or “pan-HER antibody composition” refer to a composition of antibody molecules which are capable of binding to at least two different antigens on at least two HER family receptors.
  • the individual antibodies of an antibody composition bind to different antigenic determinants of the HER family.
  • the individual antibodies of the antibody composition may thus bind to EGFR and HER2, EGFR and HER3, HER2 and HER3, or EGFR, HER2 and HER3, preferably to the three receptors EGFR, HER2 and HER3.
  • epitope is used to describe a part of a larger molecule (e.g. antigen or antigenic site) having antigenic or immunogenic activity in an animal.
  • An epitope having immunogenic activity is a portion of a larger molecule that elicits an antibody response in an animal.
  • An epitope having antigenic activity is a portion of a larger molecule to which an antibody immunospecificaliy binds as determined by any method known in the art.
  • Antigenic epitopes are not necessarily immunogenic.
  • An antigen is a substance to which an antibody or antibody fragment immunospecificaliy binds, e.g. a toxin, virus, bacteria, protein or DNA.
  • An antigen or antigenic site often has more than one epitope, unless it is very small, and is often capable of stimulating an immune response.
  • Epitopes may be linear or conformational.
  • a linear epitope generally consists of about 6 to 10 adjacent amino acids on a protein molecule that are recognized by an antibody, in contrast, a conformationai epitope consists of amino acids that are not arranged sequentially, but where an antibody recognizes a particular three- dimensional structure.
  • the amino acids forming the epitope are juxtaposed, enabling the antibody to recognize the conformationai epitope, in a denatured protein only linear epitopes are recognized.
  • a conformationai epitope by definition, must be on the outside of the folded protein.
  • distinct epitopes refers to the fact that when two different antibodies of the invention bind distinct epitopes, there is less than 100% competition for antigen binding, preferably less than 80% competition for antigen binding, more preferably less than 50% competition for antigen binding, and most preferably as little competition as possible, such as less than about 25% competition for antigen binding.
  • Antibodies capable of competing with each other for binding to the same antigen may bind the same or overlapping epitopes or may have a binding site in dose vicinity of one another, so that competition is mainly caused by steric hindrance.
  • An analysis for "distinct epitopes" of antibody pairs may be performed by methods known in the art, for example by way of binding experiments under saturating antibody conditions using either FACS (fluorescence activated cell sorting) or other flow cytometry analysis on ceils expressing the relevant receptor antigen and individual fluorescent labeled antibodies, or by Surface Plasmon Resonance (SPR) using antigen captured or conjugated to a flow cell surface.
  • FACS fluorescence activated cell sorting
  • SPR Surface Plasmon Resonance
  • the distinct epitopes are preferably "non-overlapping" in the sense that two different antibodies in a composition of the invention that bind to the same receptor have a sufficiently low competition for antigen binding that the two antibodies are able to bind their respective epitopes simultaneously, it will be understood by persons skilled in the that there can be different degrees of overlap, and that distinct epitopes can be considered to be "non- overlapping" in spite of the presence of some degree of competition, as long as the respective antibodies are able to substantially bind their epitopes. This is generally considered to be the case when the competition for antigen binding between two antibodies is less than about 50%.
  • Methods for determining competition between antibodies are known in the art, for example using Surface Plasmon Resonance (SPR) as described e.g. in WO 2011/1.07957.
  • Antibodies binding to different epitopes on the same antigen can have varying effects on the activity of the antigen to which they bind, depending on the location of the epitope.
  • An antibody binding to an epitope in an active site of the antigen may block the function of the antigen completely, whereas another antibody binding at a different epitope may have no or little effect on the activity of the antigen alone.
  • Such antibodies may, however, still activate complement and thereby result in the elimination of the antigen-expressing cell, and may result in synergistic growth inhibitory effects when combined with one or more antibodies binding at different epitopes on the same antigen.
  • the epitope is a portion of the extracellular domain of EGFR, HER2 or HERS (either wild-type or mutated) .
  • An anti-EGFR antibody of the invention will thus bind to the extracellular domain of EGFR, an anti-HER2.
  • antibody of the invention will bind to the extracellular domain of HER2, and an anti-HER3 antibody of the invention will bind to the extracellular domain of HERS.
  • compositions comprising : anti-EGFR antibody (a) and anti ⁇ HER2 antibody (c);
  • anti-EGFR antibody (a) and anti ⁇ HER2 antibody (d);
  • anti-EGFR antibody (a) and anti HERS antibody (f);
  • anti-EGFR antibody (b) and anti ⁇ HER2 antibody (d);
  • anti-EGFR antibody b
  • ant ⁇ HERS antibody e
  • anti-EGFR antibody b
  • ant HERS antibody f
  • anti-HER2 antibody c
  • anti H RS antibody f
  • anti-HER2 antibody d
  • ant -HERS antibody e
  • anti-HER2 antibodv (d) and anti -HER3 antibody (f).
  • the invention relates to a recombinant antibody composition
  • a recombinant antibody composition comprising at least one humanized anti-EGFR antibody, at least one humanized anti-HER2 antibody, and at least one humanized anti-HERS antibody.
  • the invention relates to an antibody composition
  • an antibody composition comprising at least one humanized anti-EGFR antibody, at least one humanized anti-HER2 antibody, and at least one humanized anti-HERS antibody, wherein:
  • the at least one humanized anti-EGFR antibody is selected from (a) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: l and the light chain variable region sequence of SEQ ID NQ: 2 or SEQ ID NO: 3, and (b) an antibody comprising the heavy chain variable region sequence of SEQ ID NO:4 and the light chain variable region sequence of SEQ ID NO: 5;
  • the at least one humanized anti-HER2 antibody is selected from (c) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NO: 7, and (d) an antibody comprising the heavy chain variable region sequence of SEQ ID NO:8 and the light chain variable region sequence of SEQ ID NO: 9; and
  • the at least one humanized anti-HERS antibody is selected from (e) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11, and (f) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 12 and the light chain variable region sequence of SEQ ID NO: 13.
  • the composition may thus comprise, with reference to humanized antibodies (a)-(f) outlined above:
  • anti-EGFR antibody a
  • anti-HER2 antibody c
  • anti-HER3 antibody e
  • anti-EGFR antibody (a), anti-HER2 antibody (c), and anti-HER3 antibody (f) ;
  • anti-EGFR antibody (a), anti-HER2. antibody (d), and anti-HER3 antibody (e) ;
  • anti-EGFR antibody (a), anti-HER2 antibody (d), and anti-HER3 antibody (f) ;
  • anti-EGFR antibody b
  • ants-HER2 antibody c
  • anti-HER3 antibody f
  • compositions comprising one anti-EGFR antibody, one anti-HER2 antibody and one anti-HER3 antibody are, e.g . :
  • the antibody composition comprises six humanized antibodies, i .e. two humanized antibodies directed against each of the three receptors EGFR, HER2 and HER3, where each pair of antibodies that bind the same receptor bind to distinct and non-overlapping epitopes of that receptor.
  • This may in particular be a composition comprising anti-EGFR antibodies (a) and (b), anti- HER2 antibodies (c) and (d), and anti-HER3 antibodies (e) and (f) .
  • one, two, three, four, five or all of the six antibodies may be selected from antibodies 10292, 10460, 11294, 10560, 11302, 10704, 11249, 11145, 10738, 10810, 11006 and 11052.
  • the antibody composition comprises:
  • any one or more of the antibodies (a)-(f) in this embodiment may comprise at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as " Xaa” in Table 4, e.g . substitution in up to five or six of such "Xaa” residues per antibody for one or more of the antibodies in the composition, such as substitution in one, two, three or four of such "Xaa” residues per antibody for one or more of the antibodies in the composition .
  • the antibody composition comprises anti-EGFR antibodies 11294 and 11302, anti-HER2 antibodies 11249 and 11145, and anti-H ER3 antibodies 10738 and 11052.
  • the antibody composition may comprise (a) an antibody comprising the heavy chain variable region sequence of SEQ ID NQ : 43 and the light chain variable region sequence of SEQ ID NQ :44; (b) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 47 and the light chain variable region sequence of SEQ ID NQ : 48; (c) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 51 and the light chain variable region sequence of SEQ ID NO : 52; (d) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 53 and the light chain variable region sequence of SEQ ID NO : 54; (e) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 55 and the light chain variable region sequence of SEQ ID NO : 56; and (f) an antibody comprising the heavy chain variable
  • the individual antibodies of an antibody mixture of the invention may include antibodies of more than one isotype, they may ail be of the same isotype.
  • the humanized antibodies of the invention bind to the H ER- or EGFR-famiiy members, EGFR, HER2, or H ER3.
  • HER stands for "Human Epidermal growth factor Receptor” and is often used interchangeably with the term “ErbB” to characterize the subgroup of the receptor tyrosine kinases (RTKs) consisting of the four members EGFR/ErbB, H ER2/ErbB2, H ER3/ErbB3 and H ER4/ErbB4. Together, these four receptors constitute the "HER family” (or ErbB or EGFR family) receptors.
  • RTKs receptor tyrosine kinases
  • Binding of one or more antibodies of the invention, in particular a pan-HER antibody composition of the invention, to H ER family receptors preferably inhibits the growth and proliferation of cells expressing the receptors (i .e. typically tumor cells) .
  • the mechanism (s) involved may, for example, include one or more of the following: preventing receptor dimerization, preventing ligand binding, promoting internalization and degradation of the receptor, reducing tyrosine kinase domain (TKD) phosphorylation, reducing receptor signaling, and inducing phagocytosis, CDC and/or ADCC.
  • the term "inhibits growth" is intended to include any measurable decrease in the proliferation (increase in number of cells) or metabolism of a cell when contacted with an anti-HER family antibody or pan-HER antibody composition as compared to the growth of the same cells in the absence of the antibody or composition, e.g. inhibition of growth of a ceil culture by at least about 10%, and preferably more, such as at least about 20% or 30%, more preferably at least about 40% or 50%, such as at least about 60%, 70%, 80%, 90%, 95% or 99%, or even about 100%. Growth inhibition can e.g. be determined in relevant cancer cell lines as described in the examples below,
  • the binding specificities of any two individual antibodies disclosed herein may be combined in one bispecific binding molecule.
  • a bispecific binding molecule may have the binding specificities of two antibodies targeting two different receptors selected from EGFR, HER2 and HER3, or it may have the binding specificities of two antibodies targeting the same receptor.
  • a bispecific binding molecule may have the binding specificities of anti-EGFR antibodies (a) and (b), the binding specificities of anti-HER2 antibodies (c) and (d), or the binding specificities of anti-HER3 antibodies (e) and (f). More particularly, a bispecific binding molecule may e.g.
  • the bispecific binding molecule may be a dual variable domain antibody, i.e. wherein the two arms of the antibody comprise two different variable domains, or may be in the form of an antibody fragment such as a bispecific Fab fragment or a bispecific scFv.
  • nucieic acid molecules comprising a nucleotide sequence that encodes an antibody of the invention, in particular an antibody selected from the group consisting of antibodies 10292, 10460, 11294, 10560, 11302, 10704, 11249, 11145, 10738, 10810, 11006 and 11052, or encoding a heavy and/or light chain variable region sequence of such an antibody, as well as an expression vectors comprising such a nucleic acid molecule, and host cells comprising the nucleic acid molecule or expression vector, wherein said host cells are capable of expressing an antibody encoded by the nucleic acid molecule.
  • an antibody selected from the group consisting of antibodies 10292, 10460, 11294, 10560, 11302, 10704, 11249, 11145, 10738, 10810, 11006 and 11052, or encoding a heavy and/or light chain variable region sequence of such an antibody, as well as an expression vectors comprising such a nucleic acid molecule, and host cells
  • vector refers to a nucleic acid molecule into which a nucleic acid sequence can be inserted for transport between different genetic environments and/or for expression in a host cell
  • a vector that carries regulatory elements for transcription of the nucleic acid sequence is referred to as an "an expression vector”.
  • an expression vector A vector that carries regulatory elements for transcription of the nucleic acid sequence (at least a suitable promoter) is referred to as an "an expression vector”.
  • the terms “piasmid” and “vector” may be used interchangeably.
  • Expression vectors used in the context of the present invention may be of any suitable type known in the art, e.g. a piasmid or a viral vector.
  • An additional aspect of the invention relates to methods for producing humanized recombinant antibodies and compositions comprising the antibodies of the invention.
  • One embodiment of this aspect of the invention relates to a method for producing an antibody as defined herein, comprising providing a host cell capable of expressing the antibody, cultivating said host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody.
  • the invention relates to a method for producing a recombinant antibody composition
  • a recombinant antibody composition comprising at least one humanized recombinant anti-EGFR antibody, at least one humanized recombinant anti-HER2 antibody and at least one humanized
  • the first host ceil is capable of expressing a recombinant anti-EGFR antibody of the invention
  • the second host ceil is capable of expressing a recombinant anti-HER2 antibody of the invention
  • the third host cell is capable of expressing a recombinant anti-HER3 antibody of the invention
  • cultivating the first, second and third host cells under conditions suitable for expression of the anti-EGFR antibody, the anti-HER2 antibody and the anti-HER3 antibody, and
  • An antibody or antibody composition of the present invention may be produced by methods generally known in the art for production of recombinant monoclonal or polyclonal antibodies.
  • any method known in the art for production of recombinant monoclonal antibodies may be used.
  • the individual antibodies may be produced separately, i.e. each antibody being produced in a separate bioreactor, or the individual antibodies may be produced together in single bioreactor. If the antibody composition is produced in more than one bioreactor, the purified antibody composition can be obtained by pooling the antibodies obtained from individually purified supernatants from each bioreactor.
  • compositions comprising as an active ingredient an antibody or antibody composition of the invention.
  • Such compositions are intended for amelioration, prevention and/or treatment of cancer.
  • the pharmaceutical composition may be administered to a human or to a domestic animal or pet, but will typically be administered to humans.
  • the ratio between the individual antibodies in a therapeutic composition of the invention, or, in the case of individual antibodies of the invention being administered simultaneously, sequentially or separately, will often be such that the antibodies are administered in equal amounts, but this need not necessarily be the case.
  • a composition of the invention comprising two anti-EGFR family antibodies will often contain them in approximately a 1 : 1 ratio
  • a composition comprising three anti-EGFR family antibodies will often contain them in approximately a 1 : 1 : 1 ratio
  • an antibody composition comprising six antibodies, two against each of the receptors EGFR, HER2 and HER3, will often contain them in approximately a 1 : 1 : 1 : 1 : 1 ratio.
  • Suitable ratios for the different anti-HER antibodies in compositions of the invention may be determined as described in WO 2010/040356 (incorporated herein by reference), which describes methods for identifying and selecting the optimal stoichiometric ratio between chemical entities in a combinatorial drug product, e.g. a polyclonal antibody composition, to obtain a combinatorial drug with optimal potency and efficacy.
  • the pharmaceutical composition will further comprise at least one pharmaceutically acceptable diluent, carrier or excipient.
  • pharmaceutically acceptable diluent carrier or excipient.
  • these may for example include preservatives, stabilizers, surfactants/wetting agents, emulsifying agents, solubllizers, salts for regulating the osmotic pressure and/or buffers.
  • Solutions or suspensions may further comprise viscosity- increasing substances, such as sodium carboxymethylceilulose, carboxymethylceiiulose, dextran, polyvinylpyrrolidone or gelatin.
  • a suitable pH value for the pharmaceutical composition will generally be in the range of about 5.5 to 8.5., such as about 6 to 8, e.g.
  • compositions of the invention may be prepared in a manner known per se for preparation of recombinant antibody compositions.
  • PCT/IB2011/054834 See PCT/IB2011/054834.
  • the antibodies and compositions of the present invention may be used for the treatment or amelioration of a disease in a mammal, in particular treatment of cancer in humans.
  • treatment refers to administration of an antibody or, preferably, antibody composition of the invention in a sufficient amount to ease, reduce, ameliorate or eradicate (cure) symptoms or disease states.
  • Administration of two or more pan-HER antibodies of the invention will generally be by way of simultaneous administration of the antibodies, preferably in the form of a composition containing all of the pan- HER antibodies to be used for treatment. However, it is also possible to administer two or more antibodies of the invention separately. References herein to e.g.
  • administration of a recombinant antibody composition comprising at least two anti-HER family antibodies should therefore be understood as encompassing not only administration of a composition comprising the at least two antibodies as such, but also separate administration of the antibodies. Combinations of two or more antibodies of the invention can thus be administered simultaneously, sequentially or separately.
  • One embodiment of the invention is a method of preventing, treating or ameliorating one or more symptoms associated with cancer in a human or other mammal, comprising administering an effective amount of the pharmaceutical antibody composition of the present invention to said mammal.
  • a particular embodiment relates to a method for treating a patient, typically a human patient, with a disorder characterized by expression or overexpression of or dependency on any one or more of the EGFR family receptors EGFR, HER2 and HER3, in particular cancer, the method comprising administering to said patient a recombinant antibody composition or
  • HER dependency refers to a cancer cell with dependency on one or more of the HER family receptors for maintaining malignant properties such as proliferation, growth, motility, invasion, survival and/or chemo resistance. Dependency may be caused by receptor overexpression, receptor mutations, autocrine growth factor production, and/or cross-talk with other receptor systems.
  • the invention relates to a method for treating cancer in a patient, typically a human patient, having acquired resistance to treatment with an antibody and/or a tyrosine kinase inhibitor (TKi), the method comprising administering to said patient an effective amount of a recombinant antibody composition or pharmaceutical composition as defined herein.
  • TKi tyrosine kinase inhibitor
  • an antibody composition of the invention breast, ovarian, gastric, colon, rectum, prostate, bladder, pancreas, melanoma, head and neck, and non-small cell lung cancer.
  • Antibody compositions of the invention are contemplated to be particularly applicable to treatment of cancers that overexpress EGFR or HER2, for example certain epithelial cancers such as many breast cancers, ovarian cancers and gastric (stomach) cancers.
  • antibody compositions of the invention are used to treat a patient with pancreatic cancer.
  • the patient may have a KRAS mutation.
  • the patient has not been treated for cancer previously, in another embodiment, the patient has been treated for cancer previously.
  • the patient may have been treated with cetuximab, trastuzumab, or pertuzumab previously.
  • the cancer in the patient may have acquired resistance to cetuximab, trastuzumab, or pertuzumab.
  • two main clinical pathways are contemplated, namely I) adjunctive therapy in connection with at least one additional therapeutic treatment or 2) as a monotherapy.
  • Adjunctive therapy In adjunctive therapy, also known as combination therapy, patients will be treated with antibodies of the present invention in combination with at least one additional therapeutic treatment, typically a chemotherapeutic or antineoplastic agent and/or radiation therapy.
  • the composition of the invention may also be used in combination with a different anti-cancer antibody, e.g. an antibody targeting VEGF.
  • the primary cancer targets listed above may thus be treated by administration of an antibody or composition of the invention in addition to standard first line and second line therapy. Protocol designs will address effectiveness as assessed e.g. by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. Such dosage reductions may allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent.
  • Such compounds may, for example, be selected from the group consisting of retinoic acid, trans-retinoic acids, cis- retinoic acids, phenyibutyrate, nerve growth factor, dimethyl sulfoxide, active form vitamin D3, peroxisome pro! ifera tor-activated receptor gamma, 12-O-tetradecanoylphorbol 13- acetate, hexamethylene-bis-acetamide, transforming growth factor-beta, butyric acid, cyclic AMP, and vesnarinone.
  • the compound is selected from the group consisting of retinoic acid, phertyibutyrate, a!i-trans-retinoic acid and active form vitamin D.
  • compositions of the invention comprising an antibody composition of the invention and at least one chemotherapeutic or antineoplastic compound may be used as a combination treatment for the simultaneous, separate or successive administration in cancer therapy.
  • chemotherapeutic compound may by any chemotherapeutic agent suitable for treatment of the particular cancer in question, for example an agent selected from the group consisting of alkylating agents, for example platinum derivatives such as cispiatin, carbopiatin and/or oxaliplatin; plant aikoids, for example pac!itaxe!, docetaxei and/or irinotecan; antitumor antibiotics, for example doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and/or mitomycin; ;
  • alkylating agents for example platinum derivatives such as cispiatin, carbopiatin and/or oxaliplatin
  • plant aikoids for example pac!itaxe!, docetaxei and/or irinotecan
  • antitumor antibiotics for example
  • topoisomerase inhibitors such as topotecan; and/or antimetabolites, for example fiuorouraci! and/or other fluoropyrimidines.
  • antibody composition of the invention may be used in adjunctive therapy in connection with tyrosine kinase inhibitors.
  • tyrosine kinase inhibitors synthetic, mainly quinazoline- derived, low molecular weight molecules that interact with the intracellular tyrosine kinase domain of receptors and inhibiting ligand-induced receptor phosphorylation by competing for the intracellular Mg-ATP binding site.
  • tyrosine kinase inhibitors that block HER2 kinase are currently in clinical development. Some of these also target EGFR or other EGFR family receptors.
  • Pharmaceutical articles comprising an antibody composition of the invention and at least one Ki targeting HER2 may thus also be used as a combination treatment for the simultaneous, separate or successive administration in cancer therapy.
  • the antibody compositions of the present invention may be used in combination with other antibody therapeutics, e.g. an antibody against VEGF (e.g. Avastin®).
  • the antibody compositions of the present invention may be used in combination with an agent known to stimulate cells of the immune system, such combination treatment leading to enhanced immune-mediated enhancement of the efficacy of the antibody compositions of the invention.
  • immune-stimulating agents include recombinant inter!eukins (e.g. IL-21 and IL-2).
  • the antibody composition in accordance with the present invention in monotherapy of tumors, may be administered to patients without concurrent use of a chemotherapeutic or antineoplastic agent, i.e. as a stand-alone therapy.
  • compositions of the invention are in the form of immunoconjugates, i.e. antibodies conjugated to one or more anti-cancer agents, in particular in the case of compositions of the invention that bind distinct epitopes, it is contemplated that this may generate a cross-linked antibody-receptor lattice on the ceil surface, thereby potentially resulting in an increased level of receptor internalization as compared to the use of a single monoclonal antibody.
  • Conjugation of one or more of the individual antibodies of such a composition to one or more anti-cancer agents therefore has the potential to specifically and effectively deliver the conjugated anti-cancer agents to the interior of tumor ceils, thereby augmenting the effect of the antibody composition of the invention to provide an improved tumor cell-killing activity.
  • anti-cancer agents may be conjugated to the antibodies of the invention, including cytotoxic agents (including conventional chemotherapy agents and other small molecule anti-cancer drugs), cytokines (in which case the conjugate may be termed an "immunocytokine"), toxins (in which case the conjugate may be termed an "immunotoxin”) and radionuclides, and a few immunoconjugates have already been approved for clinical use.
  • cytotoxic agents including conventional chemotherapy agents and other small molecule anti-cancer drugs
  • cytokines in which case the conjugate may be termed an "immunocytokine”
  • toxins in which case the conjugate may be termed an "immunotoxin”
  • radionuclides and a few immunoconjugates have already been approved for clinical use.
  • immunoconjugates include Zevalin® (a murine anti-CD20 antibody conjugated to 3 ⁇ 40 Y), Bexxar® (a murine anti-CD20 antibody conjugated to 131 I) and My
  • immunoconjugates that have been tested in clinical trials include antibodies conjugated to e.g. doxorubicin or a maytansinoid compound
  • immunotoxins that have been tested in clinical trials include several antibodies conjugated to a truncated Pseudomonas exotoxin A.
  • An immunocytokine comprising a humanized EpCA antibody conjugated to IL-2 has also been tested.
  • antibodies of the invention conjugated to cytotoxic agents may e.g. belong to any of the major classes of chemotherapy drugs, including alkylating agents (e.g. carboplatin, cisplatin, oxaiiplatin), antimetabolites (e.g. methotrexate, capecitabine, gemcitabine), anthracyclines (e.g. bleomycin, doxorubicin, mitornycin-C) and plant alkaloids (e.g. taxanes such as docetaxel and paciitaxei, and vinca alkaloids such as vinblastine, vincristine and vinorelbine). Since the use of immunoconjugates specifically directs the anticancer agent to the tumors, and in particular to the interior of the tumor cells subsequent to internalization, immunoconjugates based on the antibodies of the invention may
  • alkylating agents e.g. carboplatin, cisplatin, oxaiiplatin
  • antimetabolites e
  • the conjugated anti-cancer agent in an immunoconjugate is generally linked to the antibody by means of a labile linker that is relatively stable in serum but which allows release of the agent when the immunoconjugate is internalized into the target cell.
  • Suitable linkers include, for example, chemical linkers that are stable at neutral pH in serum but are subjected to acid hydrolysis in the mildly acidic conditions within the lysosomes subsequent to internalization, disulfide linkers that are cleaved by intracellular thiols, and peptide linkers that are stable in serum but which are subjected to enzymatic cleavage in intracellular compartments.
  • compositions containing two or more antibodies of the invention can be envisioned in compositions containing two or more antibodies of the invention.
  • two antibodies it would be possible to conjugate the antibodies to two or more different anti-cancer drugs or to conjugate one antibody to a prodrug which is activated by an agent such as an enzyme conjugated to the other antibody.
  • an agent such as an enzyme conjugated to the other antibody.
  • ADPT antibody-directed enzyme prodrug therapy
  • monoclonal antibodies where a prodrug is activated by an enzyme targeted to the tumor by a mAB-enzyme conjugate
  • the present invention may provide an opportunity for tailoring this approach to particular conditions, it may thus be possible to specifically increase tumor cell killing while sparing or reducing damage to normal tissues.
  • compositions of the invention comprising antibodies directed against two or more EGFR family receptors may contain a single antibody in the form of an immunoconjugate, or they may contain two or more antibodies in the form of an immunoconjugate, e.g. one or possibly two immunoconjugates targeting each of the receptors EGFR, HER2 and HER3.
  • the antibody compositions of the invention will be administered in an effective amount for treatment of the condition in question, i.e. at dosages and for periods of time necessary to achieve a desired result.
  • a therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the antibodies are being administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments.
  • An effective amount for tumor therapy may be measured by its ability to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression, e.g. by reducing tumor size.
  • the ability of an antibody or composition of the invention to inhibit cancer may be evaluated by in vitro assays, e.g. as described in the examples, as well as in suitable animal models that are predictive of the efficacy in human tumors.
  • Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.
  • an appropriate dosage of an antibody composition of the invention will be similar to the recommended dosage for the anti- HER2 monoclonal antibody trastuzumab (Herceptin®) or the anti-EGFR monoclonal antibody panitumumab (Vectibix®).
  • Herceptin® is administered (by way of infusion) for treatment of breast cancer at either an initial dose of 4 mg/kg and subsequent weekly doses of 2 mg/kg, or an initial dose of 8 mg/kg and subsequent doses of 6 mg/kg every three weeks, while Vectibix® is administered at a dose of 6 mg/kg every 14 days.
  • an antibody composition of the invention will be in the range of 0.1-100 mg/kg, such as about 0.5-50 mg/kg, e.g. about 1-20 mg/kg.
  • the antibody composition may for example be administered in a dosage of at least 0.25 mg/kg, e.g. at least 0.5 mg/kg, such as at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2 mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg, e.g. at least 5 mg/kg; and e.g. up to at most 50 mg/kg, such as up to at the most 30 mg/kg, e.g.
  • Administration will normally be repeated at suitable intervals, e.g. once every week, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the responsible doctor, who may optionally increase or decrease the dosage as necessary.
  • Intravenous deliver will presumably be the standard delivery technique for the majority of tumors.
  • intraperitoneal administration may prove favourable for obtaining high dose of antibody at the tumor and to minimize antibody clearance.
  • certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion may allow the obtainment of a high dose of the antibody at the site of a tumor and minimise short term clearance of the antibody.
  • safety concerns are related primarily to (i) cytokine release syndrome, i.e. hypotension, fever, shaking, chills, (ii) the development of an immunogenic response to the protein (i.e. development of human antibodies by the patient to the recombinant antibody product), and (Hi) toxicity to norma! cells that express the HER family receptors, e.g. many epithelial cells. Standard tests and fo!iow-up procedures are utilised to monitor any such safety concerns.
  • the antibodies of the present invention also are useful in diagnostic processes (e.g., in vitro, ex vivo).
  • the antibodies can be used to detect and/or measure the level of EGFR, HER2, or HER3 in a sample from a patient (e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, serum, bowel fluid, saliva, or urine).
  • a sample from a patient e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, serum, bowel fluid, saliva, or urine.
  • Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELiSA), chemiluminescence assays,
  • kits comprising the antibodies described herein.
  • CDR complementarity determining region
  • the CDRs of the donor murine antibodies were grafted into the closest human V-region acceptor framework, which was found by comparing the V region amino acid sequences of the donor antibodies with the human germline repertoire of V and J region sequences (i GT reference directory).
  • the closest germline V and J regions identified for each antibody are shown in Table 5 below.
  • the closest human germline V-region was IGKV2-30*02.
  • a second acceptor framework was also selected from the IGKV1 family.
  • Each of the two frameworks were used for generation of a VL back mutation library and combined with the single 1277 VH back mutation library.
  • CDR grafting alone may not be sufficient to recreate the binding specificity and affinity, and thus biological activity, of a rodent antibody
  • back mutations may have to be introduced at critical positions.
  • Potentially critical positions include those that are somatically hypermutated in the donor antibody, positions that may be in direct contact with the antigen or influencing CDR structure (structure determining residues or Vernier zone residues), positions in the VH/VL interface or responsible for the VH/VL packing angle, and positions that are occupied by statistically rare (as compared to the antibody repertoire) or structurally unfavorable residues. These positions can be identified using information available in the literature and in antibody databases (e.g., Padian (1994) Moi. Immunol.
  • Figures 1-6 Ail the hits from the screening of the initial libraries based on antibodies 1277 and
  • the chip surface was regenerated between cycles by stripping the captured antibody/antigen complexes off the surface with a 10-second injection of 3 M MgCI 2 (GE Healthcare, Denmark) repeated three times. Finally, binding responses were fitted to a Langmuir 1 : 1 binding model for calculation of the on-rate (kon or ka), off- rate (koff or kd) and affinity (KD) constants using double referencing .
  • the results of the kinetic binding analysis show that the selected variants have retained or even improved affinity for the human and cynomoigus antigen as compared to the chimeric parent antibodies (Table 6) .
  • Humanized antibody variants were tested for functional effect in a viability assay in combination with a chimeric "partner antibody” in an antibody mixture containing two antibodies against different epitopes of a particular target (where "partner antibody” refers to the fact that antibodyl277 variants (anti-EGFR) were tested together with the chimeric anti- EGFR antibody 1565, antibody 4384 variants (anti- HER2) were tested in combination with the chimeric anti-HER2 antibody 4517, and so forth) to determine if the functional synergy between the two antibodies targeting the same receptor was preserved after humanization. Each humanized variant was tested in two cell lines and compared to the parental mixture of two chimeric antibodies and to a negative control antibody.
  • partner antibody refers to the fact that antibodyl277 variants (anti-EGFR) were tested together with the chimeric anti- EGFR antibody 1565, antibody 4384 variants (anti- HER2) were tested in combination with the chimeric anti-HER2 antibody 4517, and so forth
  • ceil lines used were selected on the basis of their previously determined receptor-dependency, i .e., A431 NS epidermoid, H358 non-small cell lung , and FaDu head and neck cancers for EGFR, OE19 esophageal and BT474 breast cancer for HER2, and MDA-MB-175 VII and MCF-7 breast cancer for HER3.
  • a combination of six humanized variants (11.294, 11302, 11249, 11145, 10738 and 11052; Humanized Pan-HER) was tested in a number of cell lines and compared to the combination of the six chimeric antibodies ( 1277, 1565, 4384, 4517, 5038 and 5082; Chimeric Pan-HER) .
  • the cell lines used were selected on the basis of their previously determined dependency on the HER family receptors. Prior to performing the viabiiity assay the appropriate antibodies and antibody mixtures were diluted to a final total antibody concentration of 100 g/ml in appropriate media supplemented with 0.5-2% FBS and 1% P/S (penicillin/streptomycin), yielding a final total antibody concentration of 50 pg/mi in the well containing the highest antibody concentration.
  • a threefold serial dilution of the antibodies was then performed in a 384-weli plate, followed by addition of relevant numbers of ceils to the experimental wells.
  • the CF-7 cells were also stimulated with 1 n heregulin beta.
  • the plates were incubated for 4 days in a humidified incubator at 37°C.
  • WST-1 reagent (Roche Applied Science, Mannheim, Germany) was added to the plates and the plates were incubated for 1-3 h at 37°C. Plates were transferred to an orbital plate shaker for one h and the absorbance was measured at 450 and 620 nm
  • MAC metabolicaily active cells
  • the chimeric parent antibodies and selected humanized variants were tested for binding to EGFR, HER2 and HER3 from humans, cynomolgus monkeys and mice, as we!I as human and murine HER4, to determine whether the humanization had introduced any changes in the cross- eactivity pattern.
  • Antibody-antigen binding was measured by ELISA with coated antigens.
  • Human antigens were obtained from Sino Biologicais.
  • AH other antigens were produced in-house by transient expression in CHO or HEK293 ceils.
  • Chimeric and humanized antibodies, as well as an isotype control antibody were incubated with the coated antigens at different concentrations. After wash, bound antibodies were detected by HRP- (horse radish peroxidase)-conjugated secondary antibodies.
  • HRP- (horse radish peroxidase)-conjugated secondary antibodies were scored from negative (-; OD ⁇ 0.1) to strongly positive (+ + + ; OD>2.5).
  • a number of humanized variants of the chimeric anti-EGFR, anti-HER2 and anti-HER3 antibodies disclosed in PCT/IB2011/054834 were produced by screening of CDR-grafted libraries generated by back mutation of potentially critical framework positions and in some cases by alteration of unwanted CDR sequence motifs. Approximately 100 hits from each library selected for binding affinity to the relevant target antigen were subjected to off-rate ranking, and variants with a dissociation rate similar to or siower than that of the parent chimeric antibody were selected and sequenced . Between four and ten hits from each library screening were selected on the basis of retained or improved binding to human and cynomolgus antigen, number of back-mutations and absence of unwanted sequence motifs for larger scale expression and purification.
  • Selected purified humanized antibody variants were subjected to a kinetic binding analysis to determine binding affinity to human and cynomolgus antigen, to in vitro functional analysis in a viability assay in combination with a chimeric partner antibody binding to a different epitope of the same receptor, and to a cross-reactivity assay.
  • Each of the humanized variant antibodies 10292, 10460, 11294, 10560, 11302, 10704, 112449, 11145, 10738, 10810, 11006 and 11052 were found to exhibit functional properties that were very similar to those of the original chimeric parent antibody from which they were derived, including :
  • EXAMPLE 2 Two morsociorsai antsbodses against rsors-oveHappi g epitopes ors EGFR, HER2 or HERS display synergistic ire vitro growth inhibitory activity and effectively induce target do rs-reguiation
  • Antibodies against non-over!apping epitopes on EGFR i.e., 1277 and 1565
  • HER2 i.e., 4384 and 4517
  • HER3 i.e., 5038 and 5082
  • Antibody treatments consisted of antibodies to each receptor administered either alone or in the following combinations: 1277 and 1565 mixture, 4384 and 4517 mixture, and 5038 and 5082 mixture. Cellular damage will inevitably result in loss of the ability of the ceil to maintain and provide energy for metabolic cell function and growth.
  • Metabolic activity assays are based on this premise and usually measure mitochondrial activity.
  • the Cell Proliferation Reagent WST-1 (Roche Cat. No 11 644 807 001) is a ready-to-use substrate that measures the metabolic activity of viable cells, it is assumed that the metabolic activity correlates with the number of viable cells.
  • the WST-1 assay was used to measure the number of metaboiical!y active ceils after treatment of cancer cells with different concentrations of antibodies for 96 hours.
  • the appropriate antibodies and antibody mixes Prior to performing the WST-1 assay, the appropriate antibodies and antibody mixes were diluted to a final total antibody concentration of 100 Mg/ml in appropriate media supplemented with 2% of FBS and 1% P/S yielding a final total antibody concentration of 50 ⁇ /mi in the well containing the highest antibody concentration. A threefold serial dilution of the antibodies was then performed. Relevant numbers of cells were then added to the experimental wells in a 384-well plate. The plates were incubated for 4 days in a humidified incubator at 37°C. WST- 1 reagent was then added to the plates and the plates were incubated for one hour at 37°C.
  • MAC metaboiica!ly active cells
  • Pan-HER a mixture of six monoclonal antibodies against EGFR, HER2 and HERS; antibodies 1277, 1565, 4384, 4517, 5038, and 5082
  • antibody mixtures targeting two HER family members i .e., EGFR and HER2, EGFR and HER3, and HER2 and HER3
  • antibody mixtures targeting one HER family member i .e.
  • HNS HNS, MDA-MB175-VII, HCC827, 87, A431NS, FaDu, OE19, SW948, BT474, R G- l, TEH, GEO, H358, CALU-3, H292, HCC202, LS174T, ZR-75-30, H 1975, KYSE520, AU-565, CAPAN- 1, IGR-OV1, OE33, PK-1, CFPAC-1, BxPC3 ; A431, SW1463, COL0678, H820, COLO680N, ASPCl, HCC1937, H661 , MFE-280, OVCAR-3, OVCAR-5, SK-BR-3, SW403, OVCAR-8, RL95-2, RMUG-S, SW837, T84, CAPAN-2, GP5d, CaC02, BT2Q, MDA-MB-468, DU145, A
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR+HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR -HERS refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • E AMPLE 4 Pan-HER effectively inhibits Hig rsd-iirs lisced proisferatiors
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR-f HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Pan-Her exhibited effective- inhibition of a wide variety of cancer cell lines in the presence of EGF or Heregulin.
  • Pan-HER (a mixture of six monoclonal antibodies against EGFR, HER2 and HER3; antibodies 1277, 1565, 4384, 4517, 5038, and 5082) was tested for its ability to inhibit the growth and proliferation of HNS, OE19 and DA-MB-175-VII cell lines or cell pools with acquired resistance to cetuximab, trastuzumab, or pertuzumab, respectively.
  • Cetuximab resistant HNS cell lines were generated as described in Example 11.
  • trastuzumab resistant OE19 cells and pertuzumab resistant DA-MB-175-VII ceils were established by exposing parental ceils to increasing concentrations of trastuzumab [10- 100 pg/ml] and pertuzumab [1-50 pg/mi] respectively, during a period of eight months and 12 months respectively. Ceils were split once or twice a week in order to keep ceils in expotential growth. The level of resistance was tested every month in a WST-1 viability assay as described in Example 2, until a pool of resistant cells was established. Single cell clones of trastuzumab resistant OE1.9 cells were generated through limited dilution cloning of the acquired trastuzumab resistant pool of OE19 cells as described in Example 11.
  • EGFR, HER2 and HERB levels were measured in whole ceil lysates from H292 and OVCAR8 cell lines after antibody treatment (2.0 pg/ml) for 48 hour by western blot analysis.
  • Pan-HER a mixture of six monoclonal antibodies against EGFR, HER2 and HER3; antibodies 1277, 1565, 4384, 4517, 5038, and 5082
  • antibody mixtures targeting one HER family member i.e., EGFR
  • HER2 antibodies 4384, 4517, or 4384+4517
  • HER3 antibodies 5038, 5082, or 5038+5082
  • BxPC-3 pancreatic cancer
  • BxPC-3 pancreatic cancer cells were inoculated into mice, in brief, 5x106 BxPC3 cells were inoculated subcutaneously into the left flank of eight to ten week old female athymic nude mice. Tumors were measured thrice weekly with calipers and tumor volume in mm3 was calculated according to the formula : (width) 2 x length x 0.5. At an average tumor size of 140 mm 3 the mice were randomized and treatment initiated. The mice were treated with thrice weekly intraperitoneal injections of 50 mg/kg (10 injections in total) followed by an observation period. Graphical representations of tumor volume data were presented as means ⁇ SEM.
  • Pan-HER antibodies 1277, 1565, 4384, 451.7, 5038, and 5082
  • Pan-HER antibodies 1277, 1565, 4384, 451.7, 5038, and 5082
  • Figure 30; 7/group; treatment period indicated by the light grey area on the graph.
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR+HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+HER3 refers to a mixture of antibodies 1277., 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Pan-HER refers to a mixture of antibodies 1.277, 1565, 4384, 4517, 5038, and 5082.
  • EXAMPLE 8 Synergistic effect of targeting multiple HER family receptors in €aiu ⁇ 3 (NSCLC) xenograft modei
  • the Calu-3 (NSCLC) xenograft model were treated with antibody mixtures and the effect on tumor size assayed.
  • Calu-3 NSCLC cells were inoculated into mice.
  • 1x107 Calu-3 cells were inoculated subcutaneousiy into the left flank of eight to ten week old female athymic nude mice. Tumors were measured thrice weekly with calipers and tumor volume in mm3 was calculated according to the formula : (width)2 x length x 0.5.
  • the mice were randomized and treatment initiated. The mice were treated with thrice weekly intraperitoneal injections of 50 mg/kg (8 injections in total). Graphical representations of tumor volume data were presented as means ⁇ SEM.
  • the results show a synergistic effect of targeting EGFR, HER2 and HER3 simultaneuous!y whereas no clear synergy could be observed when targeting EGFR and HER2 or EGFR and HER3 compared to the anti-tumor response of EGFR mono-targeting.
  • EGFR refers to a mixture of antibodies 1277 and 1565.
  • HER2 refers to a mixture of antibodies 4384 and 4517.
  • HER3 refers to a mixture of antibodies 5038 and 5082.
  • EGFR+HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR+HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • E AMPLE 9 Pan-HER effectsveiy prevents compensatory receptor up-regullatsors in vivo
  • EGFR, HER2 and HERS levels were measured in antibody-treated BxPC-3 tumor iysates by Western Blot analysis.
  • Pan-HER a mixture of six monoclonal antibodies against EGFR, HER2 and HERS; antibodies 1277, 1565, 4384, 4517, 5038, and 5082
  • antibody mixtures targeting two HER family members i.e., EGFR and HER2, EGFR and HER3, and HER2 and HER3
  • antibody mixtures targeting one HER family member i.e., EGFR, HER2 and HER3
  • EGFR+HER2 refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
  • EGFR-f HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • Pan-HER is capable of effectively inducing simultaneous down-regulation of ail three targets and preventing compensatory receptor up-regulation in vivo.
  • EXAMPLE ID Synergistic effect of targeting multiple HER family receptors in patient- derived AS mutated pancreatic tumor xenograft modeis
  • EGFR+HER3 refers to a mixture of antibodies 1277, 1565, 5038, and 5082.
  • HER2+HER3 refers to a mixture of antibodies 4384, 4517, 5038, and 5082,
  • Pan-HER refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
  • Table 7 Patient-derived xenograft models of pancreatic eaocer
  • wt wiid-type
  • PD Progressive disease
  • ND Not determined.
  • E AMPLE 11 Acquired Cetuximab resistant HNS clones show decreased total levels of EGFR together with high EGFR activity
  • Cetuximab resistant HN5 clones were established by exposing parental H 5 cells to increasing concentraions of cetuximab [1-100 jjg/mi] during a period of six months. Cells were split twice a week in order to keep cells in expotential growth. The level of resistance to cetuximab was tested every month in a WST-1 viability assay as described in Example 2 until a pool of cetuximab resistant cells was established. Single ceil clones were generated through limited dilution cloning of the acquired cetuximab resistant pool of HNS cells. 0.5 cells/well were plated in 384 well plates. Growth and proliferation of single cell colonies was followed using Novartis Ceilavista Imager. The most resistant individual clones, HN5 CR2, HNS CR6, HNS CR13, and HNS CR14, were selected for further characterization ( Figure 36).
  • the binding strength of cetuximab to parental HNS and resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 was determined. Binding curves were generated by plotting fluorescence signals that were normalized to the number of ceils (DRAQ-5 staining) and cetuximab concentrations. The results demonstrate that while half-maximal binding (i.e., EC50 value) of cetuximab was unaltered, maximal binding was decreased in the resistant clones compared to parental cells ( Figure 38) .
  • the relative surface levels of EGFR were determined in parental HNS and resistant alterations HNS CR2, HNS CR6, HNS CR1.3, and HNS CR14. Briefly, ceils were stained with anti-EGFR- FITC (abeam, #11400) or an isotype control (abeam, # 18446) and the relative fluorescence of live ceils quantified by flow cytometry, The relative surface levels of EGFR were lower in cetuximab resistant HNS clones compared to the parental cells ( Figure 39) .
  • the response of cetuximab resistant alterations to EGF stimulation was tested .
  • the total levels of EGFR, levels of phosphorylated EGFR and downstream signaling molecules were determined in parental HN5 and resistant clones HNS CR2, HNS CR6, HNS CR13, and H 5 CR14.
  • Parental HNS ceils and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 were untreated or stimulated with InM EGF for 15 min before harvesting.
  • Lysates were fractionated on SDS-PAGE followed by Western Blotting for EGFR, the phosphorylated EGFR species pEGFR (Tyrl068), pEGFR(Tyrl045), pEG FR(Ty r 11 3), pEGFR(Tyr992), pEGFR(Thr669), and pEGFR(Serl046/1047), and the signalling molecules AKT, pAKT (Ser473), ERK1/2, pERKl/2(Thr202/Tyr204) ( Figures 40 and 41) .
  • ⁇ -Actin was used as a loading control .
  • Results showed that the total levels of EGFR and phosphorylated EGFR were lower in cetuximab resistant HNS clones compared to the parental cells ( Figure 40) .
  • the results also showed a decreased level of pEGFR(Serl046/1047) in the cetuximab resistant clones, indicating that the feedback mechanism regulating EGFR is less active in the cetuximab resistant clones ( Figure 40) .
  • Stimulation with EGF induced a stronger activation of pAKT and pERKl/2 in the cetuximab resistant clones compared to parental HNS cells ( Figure 41) .
  • these results demonstrate that EGFR is still active in the cetuximab resistant clones, although EGFR expression is decreased compared to parental HNS cells.
  • E AMPLE 12 Antibody mixtures targeting EGFR overcome cetisximab resistance through efficient EGFR srsterrsaii ⁇ atsori followed by degradation of the receptor
  • the levels of EGFR in cells treated with EGFR-LNA, cetuximab or EGFR-2mix for 43 hour was determined by fractionating cellular lysates on a SDS-PAGE followed by Western Blotting for EGFR. The results showed that efficient EGFR internalization followed by lysosomal degradation of the receptor was induced in antibody treated resistant cells (Figure 43), and thus providing a mechanism for the ability of the antibody mixture targeting EGFR overcome cetuximab resistance.
  • HER3 in the acquired resistance to cetuximab shown here indicates the plasticity of the RTK family as a mechanism of acquired resistance to cetuximab in vitro.
  • Antibody 1277 VH nucleotide sequence
  • Antibody 1277 VH amino acid sequence
  • Antibody 1277 light chain nucleotide sequence
  • Antibody 1277 light chain amino acid sequence
  • Antibody 1565 VH nucleotide sequence
  • Antibody 1565 VH amino acid sequence
  • Antibody 1565 light chain nucleotide sequence
  • Antibody 1565 light chain amino acid sequence
  • Antibody 4384 VH nucleotide sequence
  • tacgacttca gttggttcgt gtactggggc cagggcaccc tggtgacagt ctcg (SEQ ID NO: 22)
  • Antibody 4384 VH amino acid sequence
  • Giy Asn lie Asn Pro Ser Asn Giy Giy Thr Asn Tyr Asn Glu Lys Phe
  • Antibody 4384 light chain nucleotide sequence
  • Antibody 4384 light chain amino acid sequence
  • Antibody 4517 VH nucleotide sequence
  • Antibody 4517 VH amino acid sequence
  • Antibody 4517 light chain nucleotide sequence

Abstract

The invention relates to humanized recombinant antibodies targeting the EGFR family receptors EGFR, HER2 and HER3, compositions comprising at least one humanized anti-EGFR antibody, at least one humanized anti-HER2 antibody and at least one humanized anti-HER3 antibody, and use of the antibody compositions for treatment of cancer. The invention also relates to the use of antibodies targeting multiple EGFR-family receptors to treat cancer (e.g., pancreatic cancer) and cancer that has acquired resistance to previous therapies.

Description

HUMANIZED PAN-HER ANTIBODY COMPOSITIONS
Cross References to Other Applications
This application claims priority from U.S. Provisional Application 61/641,756, filed May 2, 2012, and from U.S. Provisional Application 61/809,159, filed April 5, 2013. The disclosures of those applications are incorporated by reference herein in their entirety.
Field of the Invention
The present invention relates to novel humanized recombinant antibodies targeting the epidermal growth factor receptor (EGFR) family and compositions comprising two or more of these antibodies for use in cancer therapy.
Background of the Invention
The epidermal growth factor receptor family (EGFR or ErbB/HER family) is a subgroup of the receptor tyrosine kinases (RTKs) and includes four members: EGFR/ErbB, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4. The members of the EGFR family are closely related single- chain modular glycoproteins with an extracellula ligand binding region, a single
transmembrane domain and an intracellular tyrosine kinase domain. In normal physiological settings the ErbB family regulates key events in coordination of cell growth, differentiation and migration. EGFR, HER2 and HER3 are believed to play crucial roles in the malignant transformation of normal ceils and in the continued growth of cancer cells. EGFR and HER2 have been found to be overexpressed by many epithelial cancers. Overexpression of EGFR and HER2 has furthermore been linked to disease progression, reduced survival, poor response and chemotherapy resistance in several human epithelial cancers. The role of HER4 in malignant transformation and cancer progression is controversial and will not be discussed further here.
EGFR and HER2 are validated cancer targets and both monoclonal antibodies and small molecule inhibitors of their tyrosine kinase have been approved for the treatment of various cancers. HERS is currently being explored as a potential therapeutic target. However, patients who initially respond to these therapies often relapse due to evolvement of acquired resistance. Pre-c!inicai research points to the involvement of the one or both of the non- targeted receptors in the development of resistance. Thus, it appears that the ErbB receptors have the ability to replace one another in order to maintain growth stimulatory signaling and a malignant phenotype. Simultaneous targeting of two or ail three receptors could therefore be a more efficient way of inhibiting cancer cells with ErbB family dependency. EGFR is a 170kDa ceil surface glycoprotein consisting of a single poiypeptide chain of 1136 amino acid residues as originally determined and described by cloning and sequencing of human cD!MAs from a human vulval carcinoma ceil line. EGFR contains three major domains: an extracellular domain, a transmembrane domain and an intracellular domain containing the tyrosine kinase. The catalytic activity of EGFR resides in the tyrosine kinase domain (residues 685-953) and is activated upon iigand binding .
The EGFR exists in two different conformations, namely a tethered conformation (closed) and an extended conformation (open) . The receptor shifts between the two conformations. In the tethered conformation domains II and IV of the extracellular region of EGFR interact, leaving the receptor in an autoinhibited state. Furthermore, domain III is held at a significant distance from domain I, whereby binding of EGF to both domains simultaneously is impossible. In the extended conformation of EGFR, domains I, II and III are stericaliy arranged in a C shape, giving room for EGF binding . Furthermore, the conformational changes induce exposure of a β- hairpin consisting of a 20 residue region in domain II, also known as the "dimerization arm". The dimerization arm extending from domain II of the EGFR makes extensive contacts with the domain II of another EGFR, thereby forming an EGFR homodimer.
Dimerization brings the active cytoplasmic tyrosine kinase domains of the receptors close enough for phosphorylation of the tyrosine residues in the regulatory regions of the receptors. Furthermore, the juxtamembrane regions of the two receptors form an antiparaliel dimer which has been found to be important in stabilizing the tyrosine kinase dimer. The "receptor- mediated" dimerization mechanism is unique for the ErbB family compared to other tyrosine kinase receptors where "ligand-mediated" dimerization is the more common theme .
A number of modes of activation of the intracellular tyrosine kinase domain of EGFR have been suggested . Unlike other receptor tyrosine kinases, the EGFR tyrosine kinase domain by default adopts a conformation normally observed only in phosphorylated and activated kinases. This indicates that the kinase domain of EGFR is constitutiveiy active. Regulation of a constitutive tyrosine kinase would thus occur through the delivery of a dimerization partner's C-terminal regulator region for trans-phosphoryiation. Another possibility is that activation of the tyrosine kinase domain involves displacement of inhibitory interactions that have not been visualized in crystallographic studies. However, crystal structure analyses of the juxtamembrane and tyrosine kinase of EGFR have revealed that an asymmetric dimer of tyrosine kinases formed upon dimerization of two EGFRs is important for regulation of the tyrosine kinase activity. In this asymmetric homodimer one of the tyrosine kinases plays the receiver while the other tyrosine kinase plays the donor. Only the receiver kinase domain has catalytic activity and proceeds to phosphoryiate tyrosine residues in the C-terminal tail of the receptor (whether in cis or trans, or both is unknown) . The clathrin-mediated endocytosis is the most important mechanism of down-regulation of EGFR, The destiny of EGFR depends on the stabiiity of the ligand-receptor complex. Upon EGF binding to EGFR the EGFR homodimer is rapidly targeted to clatbrin-coated pits and internalized through !igand-induced endocytosis. Simultaneously, EGFR is heavily ubiquitinated by the attachment of both monoubiquitin and poiyubiquitin. The ubiquitin iigase Cbi is responsible for the ubiquitination of EGFR. Cbi binds either directly or indirectly through an adaptor protein such as Grb2 to phosphorylated tyrosine residues at the regulatory region of EGFR. The binding of Cbi to EGFR via Grb2 is necessary for receptor internalization. EsplS also plays a role in EGFR internalization. The exact role of Espl5 is however still controversial. The ubiquitination is involved in endocytotic downregulation of EGFR and endosomal sorting of EGFR to lysosomes. The ubiquitin chains are recognized by the endosomal sorting complex required for transport (ESCRT) and the Hrs/STA , which retains ubiquinated proteins in the membrane of early endosomes, thereby hindering recycling of EGFR. Subsequently, EGFR is sorted into intra luminal vesicles (ILVs), which leads to delivery of EGFR to the late endosome and finally degradation In the lysosomes.
In contrast to the degradation of EGFR when bound to EGF, TGF-a binding allows receptor recycling. The TGF-a iigand dissociates rapidly from EGFR in the early endosome due to the acidic environment, leading to receptor dephosphorylation, de-ubiquitination and thereby recycling of the receptor back to the cell surface.
Human epidermal growth factor receptor 2 (HER2, ErbB2 or Neu) was first described in 1984 by Schechter et ai. HER2 consists of 1234 amino acids and is structurally similar to EGFR with an extracellular domain consisting of four subdomains I-IV, a transmembrane domain, a juxtamembrane domain, an Intracellular cytoplasmic tyrosine kinase and a regulatory C- termina! domain.
The domain Il-iV contact that restricts the domain arrangement in the tethered EGFR is absent in HER2. Three of the seven conserved residues important for stabilizing the tether in the unactivated EGFR are different in HER2. HER2 thus resembles EGFR in its extended (open) form with the dimerization arm exposed and apparently poised to drive receptor-receptor interactions. The absence of a tethered HER2 conformation indicates that the receptor lacks autoinhibition as seen for the other members of the ErbB family. A stable interface of subdomain T-III seems to keep HER2 in the extended configuration similar to the extended configuration of the EGFR-EGF complex. The interaction between domains I and ΪΙΙ involves regions corresponding to ligand-binding sites in domains ί and ΪΪΙ of EGFR, leaving no space stericaily for ligands, rendering HER2 Incapable of binding ligands. Domains II and IV form two distinct interfaces that stabilize the heterodimer formation of HER2 and another member of the ErbB family. Biophysical studies have failed to detect significant HER?, homodimerization in solution or in crystals. The residues of domain II of EGFR and HER2 are similar. However, Arg285 at the dimer interface is not conserved between EGFR and HER2. In HER2 residue 2.85 is Leu.
Mutation studies indicate that Leu at this position is partly responsible for the absence of HER2. homodimers in solution. Dimerization of intact HER2. in vivo may require additional interactions of sites in the transmembrane domain of HER2.
HER2 is the only member of the ErhB family that does not bind known !igands. HER2 is instead activated via formation of heteromeric complexes with other ErbB family members and thereby indirectly regulated by EFGR and HERS ligands. HER2 is the preferred
heterodimerization partner of the three other ErbB receptors. HER2 enhances the affinity of the other ErbB receptors for their ligands by slowing down the rate of iigand-receptor complex dissociation, whereby HER2 enhances and prolongs signaling . The ability of HER2 to enhance the iigand affinity of other ErbB receptors may reflect the promiscuous behavior of HER2 as a heterodimerization partner. Heterodimerization of HER2 and another ligand-bound receptor of the ErbB family induces cross-phosphoryiation, leading to phosphorylation of the C-terminai tyrosine residues. The most active HER2 heterodimer is the HER2-HER3 complex. HER2 complements the kinase-deficient HER3 by providing an active kinase.
In contrast to EGFR, HER2 is internalization resistant when overexpressed. Overexpression of HER2 has further been reported to inhibit endocytosis of the other ErbB family members. Two mechanisms by which HER2 escapes lysosomal degradation and thereby remains at the plasma membrane have been suggested . Either HER2 avoids internalization or it becomes efficiently recycled from endosomes back to the plasma membrane. Studies using labeled antibodies have shown that HER2 is constantly internalized and recycled. Other studies in contrast failed to identify intracellular HER2 in cells treated with compounds known to inhibit recycling .
It has been proposed that the carboxyi terminus of HER?, does not possess all signals required for internalization or that it contains an inhibitory signal essential for clathrin-mediated endocytosis. Additionally, studies have shown that HER2 heterodimers are not delivered to endosomes. A Cbi docking site like the one found on EGFR has also been identified on HER2 (Y111.2) . Cbl can thereby be recruited to HER2, leading to ubiquitination of HER2, but the actual binding efficiency of Cbl is unclear. It has been proposed that HER2 is internalization resistant due to its association with membrane protrusions. Finally, other studies have shown that the endocytosis resistance of HER2-EGFR heterodimers is associated with inefficient EGF- induced formation of clathrin-coated pits.
The third member of the ErbB family, known as human epidermal growth factor receptor 3
(HER3, ErbB3) was identified in 1989 by Kraus M. H . et al . The HERS gene encodes a protein of 1342 amino acids with striking structural similarities to EGFR and HER2. Features such as overall size, four extracellular subdomains (i-iV) with two cysteine clusters (domains II and IV), and a tyrosine kinase domain show structural similarities to EGFR and HER2. The tyrosine kinase domain of HER3 shows 59% sequence homology to the tyrosine kinase domain of EGFR.
Just iike EGFR, HER3 exists in a tethered conformation and an extended conformation, in the tethered conformation the dimerization arm is buried by interactions with domain IV, leaving domains I and HI too far apart for efficient ligand binding. Ligand binding to the extracellular domains I and HI occurs in the extended conformation of HER3 and leads to
heterodimerization with other members of the ErbB family. No HERS homodimers are formed upon ligand binding. The extended and ligand-bound HERS molecule preferentially
heterodimerizes with HER2.
In contrast to EGFR and HER2, the tyrosine kinase of HER3 has impaired catalytic activity, insufficient for any detectable biological response. Two amino acid residues which are highly conserved in the catalytic domains of protein kinases are altered in the catalytic domain of HER3. These are the substitution of aspargine for aspartic acid at residue 815 and substitution of histadine for glutamate at residue 740. The two amino acid substitutions may be the reason why HERS lacks catalytic activity of its tyrosine kinase domain. Because of the impaired intrinsic kinase activity of HER3 the receptor needs to heterodimerize with another ErbB family member in order to respond to its own ligand binding.
Little is known about endocytosis of HER3. Moreover, different studies have suggested that HER3 is endocytosis impaired to the same extent as HER2. In agreement with this, the HER3- NRG1 complex was found to be internalized less efficiently and slower than the EGFR-EGF complex, supporting the view that HER3 is not endocytosed as efficiently as EGFR. However, when the C-terminai tail of EGFR was replaced with the C-terminai tail of HER3, EGFR became endocytosis impaired, suggesting that a region in the C-terminus of HER3 protects the receptor against internalization. It has also been suggested that NRG1 does not efficiently target HER3 to degradation due to the dissociation of the ligand-receptor complexes in endosomes, as it is observed when EGF is activated by TGFa.
Targeting the ErbB family has been intensely pursued in the last decade as a cancer treatment strategy. Different treatment modalities have been explored, such as tyrosine kinase inhibitors (TKIs), monoclonal antibodies (mAbs) and !igand-traps. An advantage of monoclonal antibodies for treatment of cancer is target specificity, ensuring a low toxicity compared to conventional cytotoxic cancer chemotherapy. Monoclonal antibodies have been approved for the treatment of solid tumors with abnormally high levels of EGFR or HER2, and numerous mAbs targeting EGFR or HER2 are in clinical trials. TKIs inhibit receptor signaling by binding to the ATP-bindirtg site in the tyrosine kinase domain of EGFR and HER2. Eriotinib/Tarceva® inhibits tyrosine kinases of EGFR while lapatinib/Tykerb® inhibits tyrosine kinases of both EGFR and HER2. Both eriotinib and iaptinib are FDA approved TKis for use in the treatment of non-small iung cancer (NSCLC) and HER2 overexpressing metastatic breast cancer, respectively.
However, despite the clinical usefulness of monoclonal antibody therapy and TKis,
development of acquired resistance to the treatment is an increasing issue. Combination therapy of mAbs and conventional cytotoxic chemotherapy is one of the approaches being carried out in order to increase treatment efficacy. Furthermore, several strategies are being explored to increase the efficacy of monoclonal antibodies, including enhancement of effector functions, and direct and indirect arming of the antibodies with radionuclides or toxins.
Thus, a need exists for additional drugs to treat EGFR family-related diseases in patients, including patients who have developed resistance to existing treatments. These additional drugs also should have a low risk of provoking an undesirable immune response when used to treat human patients.
Summary of the invention
We have discovered that simultaneous targeting of two or more members of the EGFR-family (e.g., EGFR, HER2, and HERB) with humanized antibodies leads to effective inhibition of cancer growth. We have also discovered that compositions targeting multiple EGFR-family members can be used to treat cancer, such as pancreatic, bone, colon, endometrial, or urinary tract cancer, including cancer that has acquired resistance to drug therapies targeting only one EGFR-family member.
Accordingly, the present invention is directed to humanized antibodies directed against EGFR, HER2 and HER3, as well as compositions comprising two or more humanized antibodies directed against two or more of these targets. The invention is further directed to the use of the antibodies and compositions for human cancer therapy.
One aspect of the invention relates to a recombinant antibody composition comprising at least one humanized anti-EGFR antibody or an antigen-binding fragment thereof, at least one humanized anti-HER2 antibody or an antigen-binding fragment thereof, and at least one humanized anti-HER3 antibody or an antigen-binding fragment thereof.
A humanized anti-EGFR antibody of the invention may be selected from an antibody comprising the heavy chain variable region sequence of SEQ ID NO: l and the light chain variable region sequence of SEQ ID NO: 3 or SEQ ID NO: 2, and an antibody comprising the heavy chain variable region sequence of SEQ ID NO:4 and the light chain variable region sequence of SEQ ID NO: 5. in one embodiment, the anti-EGFR antibody may comprise a heavy chain variable region sequence (SEQ ID NO: l) comprising Arg44 and Val83., and a light chain variable region sequence (SEQ ID NO: 2) comprising Ala i9 and Phe92; a heavy chain variable region sequence (SEQ ID NO : l) comprising Arg44, Vai83 and Ilel04, and a light chain variable region sequence (SEQ ID NO: 3) comprising Tyr41, Leu 51 and Phe92; or a heavy chain variable region sequence (SEQ ID NO: l) comprising Arg44, Val83 and Iiel04, and a iight chain variable region sequence (SEQ ID NO: 3) comprising Leu34, Tyr41, Leu51 and Phe92. In another embodiment, the anti-EGFR antibody may comprise a heavy chain variable region sequence (SEQ ID NO:4) comprising Leu20, Iie48 and Ala68, and a Iight chain variable region sequence (SEQ ID NO : 5) comprising Val75 and Phe87; or a heavy chain variable region sequence (SEQ ID NO :4) comprising Leu20, I!e48, Leu56, and Aia68, and a light chain variable region sequence (SEQ ID NO: 5) comprising Val75 and Phe87.
In some embodiments, the invention encompasses a humanized anti -EGFR antibody whose heavy and light chain amino acid sequences comprise : SEQ ID Os:43 and 44, respectively, SEQ ID NOs : 38 and 39, respectively, SEQ ID NOs :41 and 42, respectively, SEQ ID NOs :45 and 46, respectively, or SEQ ID NQs:47 and 48, respectively, or an antigen-binding fragment thereof.
A humanized anti-HER2 antibody of the invention may be selected from an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NQ: 7, and an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 8 and the Iight chain variable region sequence of SEQ ID NO: 9. In one embodiment, the anti-HER2 antibody may comprise a heavy chain variable region sequence (SEQ ID NO: 6) comprising Ser55, Leu70, Va!72, Lys74 and Ala79, and a light chain variable region sequence (SEQ ID NO: 7) comprising Va!44, Met48 and Tyr70; or a heavy chain variable region sequence (SEQ ID NO: 6) comprising Ser55 and Val72, and a light chain variable region sequence (SEQ ID NO: 7) comprising Met48 and Tyr70. In another
embodiment, the anti-HER2 antibody may comprise a heavy chain variable region sequence (SEQ ID NO: 8) comprising Ala49, ile74 and Ser77, and a Iight chain variable region sequence (SEQ ID NO: 9) comprising Thr56, Tyr71, Ser85 and Leu 104.
In some embodiments, the invention encompasses a humanized anti-HER2 antibody whose heavy and light chain amino acid sequences comprise : SEQ ID NOs:51 and 52, respectively, SEQ ID NOs :49 and 50, respectively, or SEQ ID NOs : 53 and 54, respectively, or an antigen-binding fragment thereof.
A humanized anti-HER3 antibody of the invention may be selected from an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 10 and the Iight chain variable region sequence of SEQ ID NO: 11, and an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 12 and the light chain variable region sequence of SEQ ID NO : 13. in one embodiment, the anti-H ER3 antibody may comprise a heavy chain variable region sequence (SEQ ID NO : 10) comprising et49, Ser55 and I!e68, or Asn44, Ser55 and Thr93, and a light chain variable region sequence (SEQ ID NO : 1.1) comprising Phe36, Val44, Phe49 and Iie85, or Phe36, Phe49 and Leu73. In another embodiment, the anti-HER3 antibody may comprise a heavy chain variable region sequence (SEQ ID NO : 1.2) comprising Val46, Met49, Ser55 and Arg72, and a light chain variable region sequence (SEQ ID NO : 13) comprising Val21, Vai44 and Phe87, and optionally Thr29; or a heavy chain variable region sequence (SEQ ID NO : 12) comprising Phe41 , Val46, Met49, Ser55 and Arg72f and a light chain variable region sequence (SEQ ID NO : 13) comprising Val21 , Vai44, Tyr71, Phe87 and Leu 104,
In some em bodiments, the i nvention encompasses a hu manized anti-HER3 a ntibody whose heavy and lig ht chain a mi no acid sequences com prise : SEQ ID NOs : 55 a nd 56, respectively, SEQ ID NOs : 57 a nd 58, respectively, SEQ ID NOs : 59 a nd 60, respectively, or SEQ ID NOs : 61 a nd 62, respectively, or an a ntigen-bi nding frag ment thereof.
The invention also encompasses antibody compositions comprising two, three, four, five or six of the antibodies described above. In some embodiments, the antibody composition may comprise (i) 11294 and/or 11302; (ii) 11249 and/or 11145 ; and (iii) 10738 and/or 11052. In one embodiment, the composition comprises all six antibodies.
The antibody composition may comprise (a) anti-EGFR antibody 1.0292, 10460, or 11294; (b) anti-EGFR antibody 10560 or 11302 ; (c) anti-H ER2 antibody 10704 or 11249; (d) anti-H ER2 antibody 111.45 ; (e) anti-H ER3 antibody 10738 or 1.0810; and (f) anti-H ER3 antibody 11006 or 11052. In a preferred embodiment, the antibody composition comprises anti-EGFR antibodies 11294 and 1.1302, anti-HER2 antibodies 112.49 and 111.45, and anti-H ER3 antibodies 10738 and 11052. Antibody 10292, 1.0460, 11294, 10560, 11.302, 10704, 11249, 1 1145, 10738, 1.0810, 11006, or 11052 may comprise at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4.
In one embodiment, the antibody composition may comprise (a) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 43 and the light chain variable region sequence of SEQ ID O : 44; (b) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 47 and the light chain variable region sequence of SEQ ID NO : 48; (c) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 51 and the light chain variable region sequence of SEQ ID NO : 52; (d) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 53 and the light chain variable region sequence of SEQ ID NO : 54; (e) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 55 and the light chain variable region sequence of SEQ ID NO : 56; and (f) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 61 and the light chain variable region sequence of SEQ ID NO: 62.
Further aspects of the invention relate to a method for producing antibodies and antibody compositions of the invention; a pharmaceutical composition comprising an antibody or an antibody composition of the invention and a pharmaceutically acceptable diluent, carrier, or exdpient; a method for treating cancer in a human or other mammal comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant antibody composition or pharmaceutical composition of the invention; use of a recombinant antibody composition or a pharmaceutical composition of the invention for preparing a medicament for the treatment of cancer; and a recombinant antibody composition or pharmaceutical of the invention for use as a medicament for treatment of cancer. For human treatment, the antibodies preferably are directed to human HER family members, in some embodiments, each of these compositions comprises more than one monoclonal antibody, each binding to a different epitope in the targeted HER. In some embodiments, at least one of the antibodies is conjugated to an anti-cancer agent, e.g., a cytotoxic agent, a cytokine, a toxin, or a radionuclide.
Cancer treatable by the methods of the invention includes, without limitation, pancreatic cancer (including pancreatic cancer facilitated by a KRAS mutation), head and neck cancer, breast cancer, bone cancer, colon (including colorectal cancer) cancer, endometrial cancer, urinary tract cancer, skin cancer, lung cancer, prostate cancer, gastric cancer, esophageal cancer, ovarian cancer, other epidermal cancer, and cancers with a dependency on one or more of EGFR, HER2, and HER3.
The patient may have been treated for cancer previously. For example, the patient may have been treated with a drug targeting a single EGFR-family member and have acquired resistance to the drug (e.g., cetuximab, trastuzumab, or pertuzumab).
The invention also relates to a nucleic acid molecule comprising a nucleotide sequence encoding any of the antibody heavy or light chains or heavy or light variable regions described herein. The invention also relates to an expression vector comprising such nucleic acid molecules and a host cell comprising such nucleic acid molecules or vectors. The host cell may be capable of expressing any of the antibodies described herein.
Brief Description of the Drawings
Figure 1 : Amino acid sequence alignment of variable chains of the anti-EGFR humanized monoclonal antibodies 10292, 10460, and 11294 with the in siii'co designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT. Top: Variable heavy chains of 10292 (SEQ ID NO: 38), 10460 (SEQ ID NO:40), and 112.94 (SEQ ID NQ:42) aligned to CDR grafted seguence (1277_ CDRgrafted-H ; SEQ ID f\iO: 62) . Middle: Variable light chain of 10292. (SEQ ID NO : 39) aligned to CDR grafted sequence (1277_CDRgrafted-L; SEQ ID NQ: 63) . Bottom : Variable light chains of 1.0460 (SEQ ID NO:41 ) and 112.94 (SEQ ID NO:43) aligned to CDR grafted sequence (1277A_CDRgrafted-L; SEQ ID NQ: 64) .
Figure 2: Amino acid sequence alignment of variable chains of the anti-EGFR humanized monoclonal antibodies 10560 and 11302 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT. Top: Variable heavy chains of 10560 (SEQ ID NO:44) and 11302 (SEQ ID NO :46) aligned to CDR grafted sequence (1565.. CDRgrafted-H ; SEQ ID NO: 65) . Bottom : Variable light chains of 10560 (SEQ ID O:45) and 11302 (SEQ ID NO :47) aligned to CDR grafted sequence (1565....CDRgrafted-L; SEQ ID NQ: 66) .
Figure 3 : Amino acid sequence alignment of variable chains of the anti-HER2 humanized monoclonal antibodies 10704 and 11249 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT. Top: Variable heavy chains of 10704 (SEQ ID NO:48) and 11249 (SEQ ID NO : 50) aligned to CDR grafted sequence (4384_CDRgrafted-H ; SEQ ID NO: 67) . Bottom : Variable light chains of 10704 (SEQ ID NO:49) and 11249 (SEQ ID NO: 51) aligned to CDR grafted sequence (4384_CDRgrafted-L; SEQ ID NO: 68) .
Figure 4: Amino acid sequence alignment of variable chains of the anti-HER2 humanized monoclonal antibody 11145 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT. Top: Variable heavy chain of 11145 (SEQ ID NO: 52) aligned to CDR grafted sequence (4517_CDRgrafted-H ; SEQ ID NO: 69) . Bottom : Variable light chain of 11 145 (SEQ ID NO: 53) aligned to CDR grafted sequence (4517_CDRgrafted-L; SEQ ID NO: 70) .
Figure 5 : Amino acid sequence alignment of variable chains of the anti-HER3 humanized monoclonal antibodies 10738 and 10810 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT. Top: Variable heavy chains of 10738 (SEQ ID NO: 54) and 10810 (SEQ ID NO : 56) aligned to CDR grafted sequence (5G38.„.CDRgrafted-H ; SEQ ID NO: 71). Bottom : Variable light chains of 10738 (SEQ ID NO: 55) and 10810 (SEQ ID NO: 57) aligned to CDR grafted sequence (5038_CDRgrafted-L; SEQ ID NO: 72).
Figure 6: Amino acid sequence alignment of variable chains of the anti-HER3 humanized monoclonal antibodies 11006 and 11052 with the in siiico designed sequence made up of original murine CDRs grafted into fully human framework regions. Dots denote identity, whereas differing positions are marked with their one letter amino acid abbreviation. Shaded areas indicate CDRs as defined by IMGT. Top: Variable heavy chains of 11006 (SEQ ID NO: 58) and 11052 (SEQ ID NO: 60) aiigned to CDR grafted sequence (5082_CDRgrafted-H; SEQ ID NO: 73). Bottom : Variable light chains of 11006 (SEQ ID NO: 59) and 11052 (SEQ ID NO: 61) aiigned to CDR grafted sequence (5G82.__CDRgrafted-L; SEQ ID NO: 74),
Figure 7: In vitro activity of humanized anti-EGFR antibody variant 10292 in combination with its chimeric anti-EGFR partner antibody. A431NS cells (top panel) and H358 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 8: In vitro activity of humanized anti-EGFR antibody variant 10460 in combination with its chimeric anti-EGFR partner antibody. A431NS cells (top panel) and H358 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 9: In vitro activity of humanized anti-EGFR antibody variant 10560 in combination with its chimeric anti-EGFR partner antibody. A431NS cells (top panel) and H358 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 10: In vitro activity of humanized anti-HER2 antibody variant 10704 in combination with its chimeric anti-HER2 partner antibody. OE19 ceils (top panel) and BT474 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 11 : In vitro activity of humanized anti-HER2 antibody variant 11145 in combination with its chimeric anti-HER2 partner antibody. OE19 ceils (top panel) and BT474 cells (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 12: In vitro activity of humanized anti-HER3 antibody variant 10738 in combination with its chimeric anti-HER3 partner antibody. MBA-MD-175 VII cells (top panel) and MCF-7 cells (in the presence of 1 n heregulin beta; bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 13 : In vitro activity of humanized anti-HER3 antibody variant 10810 in combination with its chimeric anti-HER3 partner antibody. MBA-MD-175 VII ceils (top panei) and MCF-7 celis (in the presence of 1 n heregulin beta; bottom panei) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 14: In vitro activity of humanized anti-HER3 antibody variant 11006 in combination with its chimeric anti-HER3 partner antibody. MB.A-MD-175 VII ceils (top panei) and MCF-7 cells (in the presence of 1 nM heregulin beta; bottom panei) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 15 : In vitro activity of humanized anti-HER3 antibody variant 11052 in combination with its chimeric anti-HER3 partner antibody. MBA-MD-175 VII ceils (top panei) and MCF-7 celis (in the presence of 1 nM heregulin beta; bottom panei) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 16: Cross-reactivit pattern of chimeric and humanized antibodies with human, cynomolgus and murine HER family antigens. The OD signal from 40 nM antibody, measured at 450 nm using an ELISA reader, was scored from negative (■■; OD<0.1) to strongly positive ( + + + ; OD> 2.5) .
Figure 17: In vitro activity of humanized anti-EGFR antibody variant 11294 in combination with its chimeric anti-EGFR partner antibody. A431NS cells (top panel) and FaDu ceils (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 18: In vitro activity of humanized anti-EGFR antibody variant 11302 in combination with its chimeric anti-EGFR partner antibody. A431 NS ceils (top panel) and FaDu ceils (bottom panel) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 19: In vitro activity of humanized anti-HER2 antibody variant 11249 in combination with its humanized anti-HER2 partner antibody 11145. OE19 ceils (top panei) and BT474 ceils (bottom pane!) were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 20: In vitro activity of a mixture of humanized antibodies (variants 11294, 11302, 11249, 11145, 10738 and 11052; humanized Pan-HER) and a mixture of chimeric antibodies (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER). The indicated ceil lines were treated with different concentrations of the indicated antibody mixtures for 96 hours. Data are presented as means ± SEM.
Figure 21A is a schematic illustrating the interaction of Pan-HER with its EGFR (left), HER2 (middle) and HER3 (right) target proteins.
Figure 21B Is a series of charts showing the effects of treatment with EGFR (left), HER2 (middle) and HER3 (right) antibodies on the metabolic activity of A431N5, HCC202, and MDA- MB-175-VII ceil lines, respectively. The figure legend in the left panel lists from top to bottom: Negative controi, 1277, 1565, 12774- 1565. The figure legend in the center panel lists from top to bottom : Negative control, 4384, 4517, 43844-4517. The figure legend in the right panel lists from top to bottom : Negative control, 5038, 5082, 5038+5082.
Figure 21C is a series of Western blot images showing the levels EGFR (left), HER2 (middle), and HER3 (right) in the total cell lysates of A431NS, HCC202 and MDA-MB-175-VII cancer cells, respectively, that had been treated with the indicated antibodies and antibody mixtures.
Figure 22 is an image showing the receptor phosphorylation levels of EGFR (left), HER2 (middle), and HER3 (right) in 73 cancer cell lines treated with Pan -HER (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER).
Figure 23 is a table showing maximal metabolic activity as a percentage of untreated (no Heregulin or EGF) control cells (set to 100%) after treatment with Pan-HER mixture (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER), Pan-HER subcomponents and a negative controi antibody.
Figure 24 is a table showing maximal metabolic activity as a percentage of untreated control cells in the absence of ligand (set to 100%) after treatment with Pan-HER (1277, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER), Pan-HER subcomponents and a negative controi antibody in the presence of 5 nM Heregulin. Cells were exposed to medium containing antibodies and ligands for 96 hours, (i.e. ligand and antibody was added simultaneously to the cells). Figure 25 is a table showing maximal metabolic activity as a percentage of untreated control cells in the absence of ligand (set to 100%) after treatment with Pan-HER (12.77, 1565, 4384, 4517, 5038 and 5082; chimeric Pan-HER), Pan-HER subcomponents and a negative control antibody in the presence of 1 nM EGF. Ceils were exposed to medium containing antibodies and ligands for 96 hours, (i.e. iigand and antibody was added simultaneously to the ceils).
Figure 26 is an image showing the mutation status of genes listed across the top of the image of seven pancreatic cancer ceil lines (CAPAN-1, PK-1, CFPAC-1, BxPC3; ASPC1, CAPAN-2, Pan08.13f PANC-1, KP4, iaPaca-2 and PSN1).
Figure 27 is a series of graphs showing the dose-response of the CAPAN-1 cell line to Pan-HER treatment in the absence (left) or presence of Heregulin (middle) and EGF (right) ligands. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
Figure 25 is a series of graphs showing the effects of Pan-HER and reference antibodies on the metabolic activity of parental cell lines (top) and the corresponding resistant clones that have acquired resistance to cetuximab, trastuzumab or pertuzumab (bottom). "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. The figure legend in the left top panel lists from top to bottom : Pan- HER, cetuximab, Neg. control. The figure legend in the center top panel lists from top to bottom : Pan-HER, trastuzumab, Neg. control. The figure legend in the right top panel lists from top to bottom: Pan-HER, pertuzumab, Meg. control.
Figure 28 is a series of graphs showing the effects of Pan-HER and reference antibodies on the metabolic activity of parental cell lines (top) and the corresponding resistant clones that have acquired resistance to cetuximab, trastuzumab or pertuzumab (bottom). "Pan -HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. The figure legend in the left top panel lists from top to bottom : Pan- HER, cetuximab, Neg. control. The figure legend in the center top panel lists from top to bottom : Pan-HER, trastuzumab, Neg. control. The figure legend in the right top panel lists from top to bottom: Pan-HER, pertuzumab, Neg. control.
Figure 29 is a series of Western blot images showing the levels of EGFR, HER2 and HER3 in whole ceil lysates of H292 (top) and OVCAR-8 (bottom) cell lines after antibody treatment. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
Figure 30 is a graph showing the effects of treatment with Pan-HER or its subcomponents on tumor volume in the BxPC-3 xenograft model. "Pan-HER" refers to a mixture of antibodies
1277, 1565, 4384, 4517, 5038, and 5082. "EGFR" refers to a mixture of antibodies 1277 and
1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR+ HER3" refers to a mixture of antibodies 1277., 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
Figure 31 is a series of images showing EGFR and HER2 immunoiabeled sections of tumors resected from vehicle and Pan-HER treated BxPC-3 xenografts three days after withdrawal of treatment. "Pan-HER" refers to a mixture of antibodies 12.77, 1565, 4384, 4517, 5038, and 5082.
Figure 32 is a graph showing the effects of treatment with Pan-HER or its subcomponents on tumor volume in the Calu-3 xenograft model . "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HERS" refers to a mixture of antibodies 5038 and 5082. "EGFR+ HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR+ HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
Figure 33 (top) is a series of Western blot images showing the levels of EGFR, HER2, HERB and a β-actin loading control in BxPC-3 tumor iysates after antibody treatment. The relative quantification of EGFR, HER2, and HERB levels in the Western blot band intensities is shown in a series of charts in Fig . 30 (bottom) . "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+ HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR+ HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+ HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082.
Figure 34 is a series of graphs showing the effects of Pan-HER on tumor volume in ST191, ST204, ST383, STS021, ST179, ST385, STS064, ST334, STS059, and STS058 patient-derived tumor xenograft models of KRAS mutated pancreatic cancer. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
Figure 35 is a series of graphs showing the effects of treatment with Pan-HER or its subcomponents on tumor volume in ST179 and ST383 patient-derived tumor xenograft models of KRAS mutated pancreatic cancer. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. "EGFR" refers to a mixture of antibodies 1277 and 1565.
"HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+ HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR+ HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2 -HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082. Figure 36 is a schematic illustrating the development and cloning of acquired cetuximab resistant HNS clones.
Figure 37 is a graph showing the dose-response effects of cetuximab treatment on parental HNS ceils and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14.
Figure 38 is a graph showing the binding curve of cetuximab to fixed parental HNS cells and cetuximab resistant clones HN5 CR2, HNS CR6, HNS CR13, and HNS CR14.
Figure 39 is a graph showing the relative surface levels of EGFR found by fluorescence flow cytometry in parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HN5 CR13, and HNS CR14,
Figure 40 is a series of Western blot images showing the total levels of EGFR, phosphorylated EGFR species, and a β-actin loading control in cell lysates from parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 that were either untreated (left) or stimulated with EGF (right).
Figure 41 is a series of Western blot images showing the total levels of EGFR, AKT, pAKT (Ser473), ERK1/2, pERKl/2(Thr202/Tyr204), and a β-actin loading control in cell lysates from parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 that were either untreated (left) or stimulated with EGF (right).
Figure 42 is a graph showing the viability of parental HNS cells and cetuximab resistant clones HNS CR2 and HNS CR14 treated with EGFR-LNA, cetuximab, EGFR-2mix (antibodies 1277 and 1565) or controls.
Figure 43 is a series of Western blot images showing the total levels of EGFR in parental HNS cells and cetuximab resistant clones HNS CR2 and HN5 CR14 treated with EGFR-LNA, cetuximab, EGFR-2mix (antibodies 1277 and 1.565) or controls.
Figure 44 is a graph showing the viability of parental HNS cells and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 treated with the indicated EGFR antibodies. "EGFR 2mix" refers to a mixture of antibodies 1277 and 1565. "Her3 2mix" refers to a mixture of antibodies 5038 and 5082.
Figure 45 is a series of graphs showing the dose-response of parental HNS ceils (Fig. 45A) and cetuximab resistant clones HNS CR2 (Fig. 45B) viability to treatment with the indicated antibodies. "EGFR 2mix" refers to a mixture of antibodies 1277 and 1565. "Her3 2mix" refers to a mixture of antibodies 5038 and 5082. "EGFR+HER3 4mix" refers to a mixture of antibodies 1277., 1565, 5038 and 5082.
Detailed description of the invention
While some monoclonal antibodies (e.g., cetuximab, trastuzumab, and pertuzumab) have been used to treat EGFR-fami!y-related diseases, these treatments are not effective for all patients. Additionally, patients often develop resistance to such drugs after initial use. This invention is based on our discovery of new humanized antibodies targeting EGFR-famiiy members EGFR, HER2, and HER3 and that mixtures of such humanized antibodies (a humanized pan-HER antibody composition) can effectively down-regulate the targets and inhibit growth of a variety of cancer ceil lines. We have also discovered that antibody mixtures targeting EGFR-famiiy members EGFR, HER2, and HER3 effectively suppress tumor growth in multiple xenograft models of human cancer, including hard-to-treat patient-derived models of pancreatic cancer. We have also shown that antibody mixtures targeting more than one EGFR-famiiy member retain their inhibitory effect in cells that have acquired resistance to therapeutic monoclonal antibodies such as cetuximab, trastuzumab, and pertuzumab.
Humanized Antibodies
One aspect of the invention relates to humanized antibodies that bind the EGFR-famiiy members EGFR, HER2, and HER3. The term "antibody" or "antibody molecule" describes a functional component of serum and is often referred to either as a collection of molecules
(antibodies or immunoglobulin) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody is capable of binding to or reacting with a specific antigenic
determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody is usually regarded as
monospecific, and a composition of antibodies may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of two or more different antibodies reacting with the same or different epitopes on the same antigen or even on distinct, different antigens). Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, and ail natural antibodies have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known collectively as immunoglobulins.
Unless otherwise indicated, the terms "antibody" or "antibodies" as used herein are intended to include single chain antibodies as well as binding fragments of antibodies, such as Fab, F(ab')2, Fv fragments or single chain Fv (scFv) fragments, and multimeric forms such as dimeric igA molecules or pentavalent IgM. In the present description and claims, references to an "antibody" or "antibodies" are therefore intended to encompass, in particular, binding fragments and single chain antibodies, unless it is indicated otherwise or apparent from the context that this is not the case.
Each heavy chain of an antibody typically includes a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region typically includes three domains, referred to as CHI, CH2 and CHS. Each antibody light chain typically includes a light chain variable region (VL) and a light chain constant region. The light chain constant region typically includes a single domain, referred to as CL. The VH and VL regions may be further subdivided into regions of hypervariability ("hypervariable regions", which may be
bypervariable in sequence and/or in structurally defined loops). The "hypervariable" regions found in the variable domains of an antibody that are primarily responsible for determining the antibody's binding specificity. These are also referred to as complementarity determining regions (CDRs), which are interspersed with regions that are more conserved, termed framework regions (FRs) . Each of the heavy and light chains of an antibody contains three CDR regions, referred to as CDRi, CDR2 and CDRS, of which CDRS snows the greatest variability. Each VH and VL typically includes three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FRi, CDRI, FR2, CDR2, FRS, CDRS, FR4. The amino acid residues in the variable regions are often numbered using a standardized numbering method known as the Kabat numbering scheme (Kabat et ai. (1991) Sequences of Proteins of Immunological interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, USA), although other numbering schemes such as Chothia and IMGT also exist.
The term "recombinant antibody" refers to an antibody that is expressed from a ceil or ceil line transfected with an expression vector (or possibly more than one expression vector, e.g. two expression vectors) comprising the coding sequence of the antibody, where said coding sequence is not naturally associated with the cell.
The four-digit antibody numbers used herein, i.e. 1277, 1565, 4384, 451.7, 5038 and 5082, refer to the chimeric parent antibodies disclosed in WO 2012/059857, from which the humanized antibodies of the invention are derived. Table 1 below shows the SEQ ID NOs, as set forth in Table 8, for the DNA and amino acid sequences of the heavy chain variable regions (VH) and the light chains (LC) of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. TabUe 1: SEQ ID NOs for the DMA and amino acid sequences of the heavy chairs variable re io s and li ht chains of chimeric antibodies
Figure imgf000021_0001
The specificity of an antibody's interaction with a target antigen resides primarily in the amino acid residues located in the six CDRs of the heavy and light chain. The amino acid sequences within CDRs are therefore much more variable between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally any specific antibody with a given amino acid sequence, by constructing expression vectors that express CDR sequences from the specific antibody grafted into framework sequences from a different antibody. As a result, it is possible to "humanize" a non- human antibody and still substantially maintain the binding specificity and affinity of the original antibody. A more detailed discussion of humanization is provided below.
A "chimeric antibody" refers in its broadest sense to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically an antibody that is partially of human origin and partially of non-human origin, i .e. derived in part from a non-human animal, for example a mouse, rat or other rodent, or an avian such as a chicken. Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of a human anti-antibody response, e.g . a human anti-mouse antibody response in the case of a murine antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine sequences derived from immunization of a mouse, while the constant region sequences are human. In the case of a chimeric antibody, the non-human parts may be subjected to further alteration in order to humanize the antibody. As described elsewhere herein, the present invention is based on humanization of certain chimeric antibodies having murine variable region sequences.
The term "humanize" refers to the fact that where an antibody is wholly or partially of non- human origin, for example a murine antibody obtained from immunization of mice with an antigen of interest or a chimeric antibody based on such a murine antibody, it is possible to replace certain amino acids, in particular in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize an immune response in humans, it is known that ail antibodies have the potential for eliciting a human anti-antibody response, which correlates to some extent with the degree of "humanness" of the antibody in question. Although it is not possible to precisely predict the immunogenicity and thereby the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic than human antibodies. Chimeric antibodies, where the foreign (usually rodent) constant regions have been replaced with sequences of human origin, have been shown to be generally less immunogenic than antibodies of fully foreign origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies. For chimeric antibodies or other antibodies of non-human origin, it is therefore preferred that they be humanized to reduce the risk of a human anti-antibody response.
For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of a complementarity determining regions (CDRs) will most often not be altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a giycosylation site, a deamidation site, an aspartate isomerization site or an undesired cysteine or methionine residue. N-linked giycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N-glycosyiation site may be achieved by mutating either the Asn or the Ser/Thr residue to a different residue, preferably by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Aia. When such a deamidation site, in particular Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues.
Numerous methods for humanization of an antibody sequence are known in the art; see e.g. the review by Almagro & Fransson (2008) Front Biosci. 13 : 1619-1633. One commonly used method is CDR grafting, which for e.g. a murine-derived chimeric antibody involves identification of human germline gene counterparts to the murine variable region genes and grafting of the murine CDR sequences into this framework. CDR grafting may be based on the
Kabat CDR definitions, although a more recent publication ( agdeiaine-Beuzeiin et ai. (2007)
Crit Rev. Oncol Hematol. 64 : 2.10-225) has suggested that the IMGT® definition (the international Im unoGeneTics information system®, www.imgt.org) may improve the result of the humanization (see Lefranc et ai. (2003), IMGT unique numbering for immunoglobulin and T ceil receptor variable domains and Ig superfamily V-!ike domains, Dev. Comp Immunol.
27, 55-77). Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR-grafted non-human antibody, back mutations (sometimes referred to as "framework repair") may be introduced at selected positions of the CDR-grafted antibody, typically in the framework regions, in order to reestablish the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molecule, while residues that are buried or that have a low degree of surface exposure will not normally be altered . An alternative humanlzation technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non- human origin are retained, while surface residues are altered to human residues.
In certain cases, it may also be desirable to alter one or more CDR amino acid residues in order to improve binding affinity to the target epitope. This is known as "affinity maturation" and may optionally be performed in connection with humanlzation, for example in situations where humanization of an antibody leads to reduced binding specificity or affinity and it is not possible to sufficiently improve the binding specificity or affinity by back mutations alone. Various affinity maturation methods are known in the art, for example the in vitro scanning saturation mutagenesis method described by Burks et al . (1997) PNAS USA, vol . 94, pp. 412- 417 and the stepwise in vitro affinity maturation method of Wu et al . (1998) PNAS USA, vol . 95, pp. 6037-6042.
Amino acid residues herein may be indicated by either the one-letter code or the three-letter code. Amino acid substitutions relative to a reference sequence may e.g . be indicated using the format "G44R", which indicates that a glycine residue in position 44 of a reference sequence has been mutated to an arginine residue. For example, in Table 2 below, "G44R" indicates a mutation of the glycine residue in a CDR-grafted antibody to an arginine residue. An amino acid residue written in the format "Arg44" indicates a particular residue in a particular position, i.e. in this case an arginine residue in position 44. Unless otherwise indicated, numbering of amino acid residues refers to the appended sequence listing.
As noted above, the present invention relates to humanized antibodies, more particularly to humanized antibodies based on certain chimeric parent antibodies described in WO
2012/059857. The humanized antibodies of the invention were developed using CDR grafting and back mutations, and in some cases alteration of unwanted sequence motifs, starting with selected chimeric anti-EGFR, anti-HER2 and anti-HER3 antibodies described in WO
2012/059857. The particular methods used to develop these humanized antibodies, as well as the results of functional evaluation of the humanized antibodies compared to the original chimeric antibodies from which they were developed, are described in the examples below.
Strikingly, the data presented in the examples shows that mixtures containing a humanized antibody of the invention have an in vitro efficacy that is comparable to that of corresponding mixtures of the original chimeric antibodies, demonstrating that the humanization process did not affect the inhibitory properties of these antibodies or their ability to function in
combination with each other. The data also strongly suggests that the humanized antibody mixtures will also show an in vivo efficacy that is comparable to that of the original chimeric antibody mixtures described in WO 2012./059857.
The five-digit antibody numbers used herein, e.g. "antibody 10560", refer to the specific humanized antibodies described below, which have been prepared by CDR grafting based on a chimeric parent antibody. For example, antibody 10560 is an antibody with a heavy chain comprising the heavy chain variable region sequence (VH) set forth in SEQ ID NO:4 and a light chain comprising the light chain variable region sequence (VL) set forth in SEQ ID NO: 5, and comprising substitutions (for example, back mutations) at certain positions compared to the original CDR-grafted antibody (see Table 3 and Figures 1-6). In the examples below, the antibodies also included a human kappa constant region sequence (SEQ ID NO:42 in WO 2012/059858 and US 2011/0217305, with an N-terminai Arg residue) and a human IGHG1 heavy chain constant region sequence (SEQ ID NO:44 in WO 2012/059858 and US
2011/0217305) .
Particular humanized antibodies of the invention are described herein by way of an antibody number, i.e. 10292, 10460, 11294, 10560, 10704, 11302, 11145, 11249, 10738, 10810, 11006 or 11052. These are derived from the chimeric antibodies (murine variabie regions, human constant regions) disclosed in WO 2012/059857 by CDR grafting and subsequent mutation at certain positions, primarily back mutations, as described in Example 1. Table 2 below outlines how the humanized antibodies of the invention are related to the chimeric parent antibodies disclosed in WO 2012/059857.
Table 2; Huma ized and chimeric parent antibody umbers
Figure imgf000024_0001
Table 3 below provides the SEQ ID NOs of exemplary humanized antibodies of the Invention, as well as the individual substitutions (back mutations, and in certain cases mutation(s) to alter undesired sequence motifs) in the heavy chain (HC) and light chain (LC) compared to the original CDR-grafted antibody. The amino acid sequences of the heavy and light chains of the antibodies listed in Table 3 are provided in Figures 1-6 and in separate SEQ ID NOs enclosed in parentheses in Tabie 3. The CDR sequences in Figures 1-6 are indicated with shading.
Table 3; Sequence numbers and substitutions m selected humanized antibodies
Figure imgf000025_0001
An indication that any of the numbered humanized antibodies listed in Table 2 may comprise "at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4" means that the antibodies may comprise additional substitutions in one or more "Xaa" residues other than the substitutions listed above in Table 3.
TabUe 4; Sequences of selected human ze antibodies
SEQ ID NO:l
<210> 1
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 1277 VH
<220>
<221> VARIANT
<222> (44) .. (44)
<223> Xaa - Gly or Arg
<220>
<221> VARIA T
<222> (49) .. (49)
<223> Xaa = Ser or Ala.
<220>
<221> VARIANT
<222> (83) .. (83)
<223> Xaa. - Met or Val
<220>
<221> VARIANT
<222> (104) . , (104)
<223> Xaa - Met or He
<400> 1
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Tyr Ser
20 25 30
Asp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Xaa Leu Glu Trp Val
35 40 45
Xaa Tyr Met Ser Ser Ala Gly Asp Val Thr Phe Tyr Ser Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gin Xaa Asn Ser Leu Arg Ala. Glu Asp Thr Ala. Val Tyr Tyr Cys
85 90 95
Val Arg His Arg Asp Val Ala Xaa Asp Tyr Trp Gly Gin Gly Thr Thr
100 105 110 Val Thr Val Ssr Ser
115
SEQ ID NO: 2
<210> 2
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 1277 VL
<220>
<221> VARIANT
<222> (13) .. (13)
<223> Xaa = Ala o Val
<220>
<221> VARIANT
<222> (19) .. (19)
<223> Xaa - Val or Ala
<220>
<221> VARIANT
<222> (33) .. (33)
<223> Xaa can be any na rally occurring amino acid
<220>
<221> VARIANT
<222> (34) .. (34)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> VARIANT
<222> (42) .. (42)
<223> Xaa = Gin or Leu
<220>
<221> VARIANT
<222> (48) .. (48)
<223> Xaa - Ala or Ser
<220>
<221> VARIANT
<222> (83) .. (83)
<223> Xaa = Leu or Val
<220>
<221> VARIANT
<222> (89) .. (89)
<223> Xaa - Ala or Gly
<220>
<221> VARI NT
<222> (92) .. (92)
<223> Xaa = Tyr or Phe
<220>
<221> VARIANT
<222> (108) . , (108)
Figure imgf000028_0001
<220>
<221> VARIANT
<222> (51) .. (51)
<223> Xaa = Arg o Leu
<220>
<221> VARIANT
<222> (92) .. (92)
<223> Xaa = Tyr or Phe
<220>
<221> VARIANT
<222> (108) .. (108)
<223> Xaa = Val or Leu
<400> 3
Asp Val Val Met. Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15
.n Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser
20 25 30
Xaa Xaa Asn Thr Tyr Leu His Trp Xaa Xaa Gin Arg Pro Gly Gin Sei
35 40 45
Pro Arg Xaa Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Xaa Cys Ser Gin Ser
85 90 95
Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Xaa Glu lie Lys
100 105 110
SEQ ID NO:
<210> 4
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 1565 VH
<220>
<221> VARIANT
<222> (20) .. (20)
<223> Xaa = Val or Leu
<220>
<221> VARIANT <222> (38) .. (38)
<223> Xaa = Arg
<220>
<221> VARIANT
<222> (40) .. (40)
<223> Xaa - Ala or Arg
<220>
<221> VARIANT
<222> (48) .. (48)
<223> Xaa - Met or He
<220>
<221> VARIANT
<222> (55) .. (55)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> VARIANT
<222> (56) .. (56)
<223> Xaa can be any naturally occurrincj amino acid
<220>
<221> VARIANT
<222> (68) .. (68)
<223> Xaa = Val or Ala
<220>
<221> VARIANT
<222> (70) .. (70)
<223> Xaa - Met or Leu
<220>
<221> VARIANT
<222> (72) .. (72)
<223> Xaa = Arg or Val
<220>
<221> VARIANT
<222> (74) .. (74)
<223> Xaa - Thr or Lys
<220>
<221> VARIANT
<222> (79) .. (79)
<223> Xaa = Val or Ala
<400> 4
Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Gin Trp Val Xaa Gin Xaa Pro Gly Gin Gly Leu Glu Trp Xaa
35 40 45
Figure imgf000031_0001
50 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Xaa Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp Phe Ala. Thr Tyr Xaa Cys Gin Gin Tyr Ser Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu He Lys
100 105
SEQ ID NO: 6
<210> 6
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 4384 VH
<220>
<221> VARI NT
<222> (38) .. (38)
<223> Xaa = Arg or Lys
<220>
<221> VARIANT
<222> (48) .. (48)
<223> Xaa - Met or He
<220>
<221> VARIANT
<222> (55) .. (55)
<223> Xaa = Asn or Ser
<220>
<221> VARIANT
<222> (68) .. (68)
<223> Xaa - Val or Ala
<220>
<221> VARIANT
<222> (70) .. (70)
<223> Xaa = Met or Leu
<220>
<221> VARIANT
<222> (72) .. (72)
<223> Xaa - Arg or Val
<220>
<221> VARIANT
<222> (74) .. (74)
<223> Xaa = Thr or Lys
<220>
<221> VARIANT <222> (79) .. (79)
<223> Xaa - Val or Ala
<400> 6
Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His
20 25 30
Trp Met His Trp Val Xaa Gin Ala Pro Gly Gin Gly Leu Glu Trp Xaa
35 40 45
Gly Asn lie Asn Pro Ser Xaa Gl Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60
Lys Ser Arg Xast Thr Xast Thr Xast Asp Xast Ser Thr Ser Thr Xa a 'i'y.i 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Arg Ala Tyr Tyr Asp Phe Ser Trp Phe Val Tyr Trp Gly Gin Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
SEQ ID NO : ?
<210> 7
<211> 106
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 4384 VL
<220>
<221> VARIANT
<222> (44) .. (44)
<223> Xaa = Pro or Val
<220>
<221> VARIANT
<222> (48) .. (48)
<223> Xaa = lie or Met
<220>
<221> VARIANT
<222> (70) .. (70)
<223> Xaa ~ Phe or Tyr <220>
<221> VARIANT
<222> (72) .. (72)
<223> Xaa = Phe or Leu
<220>
<221> VARIANT
<222> (86) .. (86)
<223> Xaa = Tyr or Phe
<400> 7
Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr He Thr Cys Arg Ser Ser Gin Asp He Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Xaa Lys Leu Leu Xaa
35 40 45
He Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Xaa Thr Xaa Thr He Ser Ser Leu Gin Pro Glu 65 70 75 80
Asp He Ala Thr Tyr Xaa Cys Gin Gin Gly Asn hr Leu Pro Leu Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu He Lys
100 105
SEQ ID NO: 8
<210> 8
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 4517 VH
<220>
<221> VARIANT
<222> (39) .. (39)
<223> Xaa - Gin or Leu
<220>
<221> VARI NT
<222> (40) .. (40)
<223> Xaa = Ala or Thr
<220>
<221> VAR.IANT
Figure imgf000035_0001
<220>
<221> VARIANT
<222> (13) .. (13)
<223> Xaa = Ala or Val
<220>
<221> VARIANT
<222> (48) .. (48)
<223> Xaa - He or Val
<220>
<221> VARIANT
<222> (56) .. (56)
<223> Xaa = Asp or Thr
<220>
<221> VARIANT
<222> (71) .. (71)
<223> Xaa = Phe or Tyr
<220>
<221> VARIANT
<222> (84) .. (84)
<223> Xaa = Ala or Gly
<220>
<221> VARIANT
<222> (85) .. (85)
<223> Xaa = Thr or Ser
<220>
<221> VARIANT
<222> (104) .. (104)
<223> Xaa = Val or Leu
<400> 9
Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Xaa Ser Val Gly 1 5 10 15
Asp Arg Val Thr He Thr Cys Arg Ala Ser Glu Asn He Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Xaa
35 40 45
Tyr Ala Ala Thr Asn Leu Ala Xaa Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Xaa Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp Phe Xaa Xaa Tyr Tyr Cys Gin His Phe Trp Gly Thr Pro Trp
85 90 95 Thr Phe Gly Gin Gly Thr Lys Xaa Glu lie
100 105
SEQ ID NO: 10
<210> 10
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 5038 VH
<220>
<221> VARIANT
<222> (44) .. (44)
<223> Xaa = Lys or Asn
<220>
<221> VARIANT
<222> (49) .. (49)
<223> Xaa - He or Met
<220>
<221> VARIA
<222> (55) .. (55)
<223> Xaa = Asp or Ser
<220>
<221> VARIANT
<222> (68) .. (68)
<223> Xaa - Val or He
<220>
<221> VARIANT
<222> (72) .. (72)
<223> Xaa - Val or Arg
<220>
<221> VARIANT
<222> (93) .. (93)
<223> Xaa - Val or Thr
<400> 10
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro
1 5 10
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr Ser Gly
20 25 30
Phe Tyr Trp Thr Trp lie Arg Gin His Pro Gly Xaa Gly Leu Glu Trp
35 40 45
Xaa Gly Phe He Ser Tyr Xaa Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60
Figure imgf000038_0001
<400> 11
Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr lie Thr Cys Arg Pro Ser Gin Asp lie Ser Asn Tyr
20 25 30
Val Asn Trp Xaa Gin Gin Lys Pro Gly L s Ala Xaa Lys Leu Leu He
35 40 45
Xaa His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Xaa Thr Xaa Thr He Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp He Ala Xaa Tyr Xaa Cys Gin Gin Gly He Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys
100 105
SEQ ID NO: 12
<210> 12
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> Humanized 5082 VH
<220>
<221> VARIANT
<222> (41) .. (41)
<223> Xaa - His or Phe
<220>
<221> VARIANT
<222> (46) .. (46)
<223> Xaa = Leu or Val
<220>
<221> VARIANT
<222> (49) .. (49)
<223> Xaa - He or Met
<220>
<221> VARIANT
<222> (55) .. (55)
<223> Xaa = Asp or Ser
<220> <221> VARIANT
<222> (68) .. (68)
<223> Xaa - Val or He
<220>
<221> VARIA T
<222> (72) .. (72)
<223> Xaa = Val or Arg
<220>
<221> VARIANT
<222> (86) .. (86)
<223> Xaa. - Val or Leu
<400> 12
Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val L s Pro Ser Gin 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser He Thr Ser Ala
20 25 30
Tyr Tyr Trp Asn Trp He Arg Gin Xaa Pro Gly Lys Gly Xaa Glu Trp
35 40 45
Xaa Gly Tyr He Gly Tyr Xaa Gly Arg Asn Thr Tyr Asn Pro
50 55 60
Lys Asn Arg Xaa Thr lie Ser Xaa Asp 'i'hr Ser Lys Asn Gin Pb.e Ser 65 70 75 80
Leu Lys Leu Ser Ser Xaa. 'i'hr Ala. Ala Asp 'i'hr Ala. Val Tyr Tyr Cys
85 90 95
Ser Arg Glu Gly Asp Tyr Gly Tyr Ser Asp Tyr Trp Gly Gin Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
SEQ ID NO: 13
<210> 13
<2H> 107
<212> PRT
<213> Artificial Seque
<220>
<223> Humanized 5082 V
<220>
<221> VARIANT
<222> (21) .. (2i; <223> Xaa - He or Val
<220>
<221> VARIANT
<222> (29) .. (29)
<223> Xaa - He or Thr
<220>
<221> VARIANT
<222> (44) .. (44)
<223> Xaa - Pro or Val
<220>
<221> VARIANT
<222> (69) .. (69)
<223> Xaa - Thr or He
<220>
<221> VARIANT
<222> (71) .. (71)
<223> Xaa - Phe or Tyr
<220>
<221> VARIANT
<222> (87) .. (87)
<223> Xaa - Tyr or Phe
<220>
<221> VARIANT
<222> (104) .. (104)
<223> Xaa - Val or Leu
<400> 13
Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Xaa Thr Cys Arg Ala Ser Gin Asp Xaa Asn Asn Tyr
20 25 30
Leu Asn Trp xyr Gi Gin Lys Pro Gly Lys Ala Xaa Lys Leu L&U lie
35 40 45
Tyr Tyr Thr Ser Arg Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Xaa Asp Xaa Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Xaa Cys Gin Gin Ser Glu Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gin Gly Thr Lys Xaa Glu He Lys
100 105 Amino acid sequence alignments of the CDR-grafted heavy and light chain variable regions of these humanized antibodies with the respective in si!ico designed sequence made up of original murine CDRs grafted into fully human framework regions are shown in Figures 1-6.
One aspect of the invention relates to particular humanized antibodies targeting EGFR., HER2. or HER3, These individual antibodies include the following :
® (a) a humanized anti-EGFR antibody comprising the heavy chain variable region
sequence of SEQ ID NO: l and the light chain variable region sequence of SEQ ID NO : 2 or SEQ ID NO:3;
® (b) a humanized anti-EGFR antibody comprising the heavy chain variable region
sequence of SEQ ID NO:4 and the light chain variable region sequence of SEQ ID NO: 5;
* (c) a humanized anti-HER2. antibody comprising the heavy chain variable region
sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NO: 7;
* (d) a humanized anti-HER2 antibody comprising the heavy chain variable region
sequence of SEQ ID NO:8 and the light chain variable region sequence of SEQ ID IMO:9;
* (e) a humanized anti-HER3 antibody comprising the heavy chain variable region
sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: ll ; and
* (f) a humanized anti-HER3 antibody comprising the heavy chain variable region
sequence of SEQ ID NO: 12 and the light chain variable region sequence of SEQ ID NO: 13.
The above-outlined humanized antibodies typically include, in both the heavy chain variable region sequence and the light chain variable region sequence, one or more of the possible substitutions (primarily back mutations, but in certain cases also mutation to alter unwanted sequence motifs) set forth in Table 4 and in the examples and accompanying figures. The heavy chain variable region sequence and the light chain variable region sequence will typically each comprise two, three, four or five such substitutions.
Examples of a preferred anti-EGFR antibody (a) are antibodies comprising:
(i) a heavy chain variable region sequence (SEQ ID NO: l) comprising Arg44 and Vai83, and a light chain variable region sequence (SEQ ID NO: 2) comprising Aial9 and Phe92. [e.g., antibody 10292] ; (ii) a heavy chain variabie region sequence (SEQ ID NO: i) comprising Arg44, Vai83 and Ilel04, and a light chain variabie region sequence (SEQ ID NO: 3) comprising Tyr41, Leu 51 and Phe92 [e.g.., antibody 10460] ; or
(iii) a heavy chain variabie region sequence (SEQ ID NO: 1) comprising Arg44, Vai83 and Iiel04, and a iight chain variabie region sequence (SEQ ID NO: 3) comprising Leu34, Tyr41, Leu 51 and Phe92 [e.g., antibody 11294] .
The anti-EGFR antibody (a) may also be an antibody corresponding to antibody 10292, 10460, or 11294, but comprising at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4, e.g. substitution in one, two, three or four of such "Xaa" residues. SEQ ID NO: 2 includes Xaa in positions 33-34, since the CDR-grafted sequence has a deamidation site (Asn-Gly) in these positions. Although it is possible to perform substitutions in both positions, it is sufficient to alter only one of the two positions in order to eliminate the deamidation site. The sequence will therefore typically include either Asn in position 33 or Gly in position 34.
An example of a preferred anti-EGFR antibody (b) is one comprising:
(i) a heavy chain variable region sequence (SEQ ID NO:4) comprising Leu20, I!e48 and Aia68, and a light chain variable region sequence (SEQ ID NO: 5) comprising Val75 and Phe87 [e.g., antibody 10560] ; or
(ii) a heavy chain variable region sequence (SEQ ID NO:4) comprising Leu20, I!e48, Leu56, and Ala68, and a iight chain variabie region sequence (SEQ ID NO: 5) comprising Val75 and Phe87 [e.g., antibody 11302] .
The anti -EGFR antibody (b) may also be an antibody corresponding to antibody 10560 or 11302, but comprising at least one additional substitution in any of the heavy chain and/or Iight chain amino acid residues indicated as "Xaa" in Table 4, e.g. substitution in one, two, three or four of such "Xaa" residues. SEQ ID NO:4 includes Xaa in positions 55-56, since the CDR-grafted sequence has a deamidation site (Asn-Gly) in these positions. Although it is possible to perform substitutions in both positions, it is sufficient to alter only one of the positions in order to eliminate the deamidation site. The sequence will therefore typically include either Asn in position 55 or Gly in position 56.
An example of a preferred anti-HER2 antibody (c) is one comprising:
(i) a heavy chain variable region sequence (SEQ ID NO: 6) comprising Ser55, Leu70, Vai72, Lys74 and Ala79, and a iight chain variabie region sequence (SEQ ID NO: 7) comprising Vai44, Met48 and Tyr70 [e.g., antibody 10704] ; or
(ii) a heavy chain variabie region sequence (SEQ ID NO: 6) comprising Ser55 and Vai72, and a Iight chain variable region sequence (SEQ ID NO: 7) comprising Met48 and Tyr70 [e.g., antibody 11249] . The anti-HER2 antibody (c) may also be an antibody corresponding to antibody 10704 or 1.1249, but comprising at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4, e.g. substitution in one, two, three or four of such "Xaa" residues.
An example of a preferred anti-HER2 antibody (d) is one comprising a heavy chain variable region sequence (SEQ ID NO:8) comprising Aia49, Iie74 and Ser77, and a light chain variable region sequence (SEQ ID NO:9) comprising Thr56, Tyr71, Ser85 and Leul04 [e.g., antibody 11145] . The anti-HER2 antibody (d) may also be an antibody corresponding to antibody 11145, but comprising at ieast one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4, e.g. substitution in one, two, three or four of such "Xaa" residues.
Examples of a preferred anti-HER3 antibody (e) are antibodies comprising a heavy chain variable region sequence (SEQ ID NO: 10) comprising et49, Ser55 and Iie68, or comprising Asn44, Ser55 and Thr93, and a light chain variable region sequence (SEQ ID NO: 11) comprising Phe36, Val44, Phe49 and Iie85, or comprising Phe36, Phe49 and Leu73. Particular examples of such anti-HER3 antibodies are those comprising :
(i) a heavy chain variable region sequence (SEQ ID NO: 10) comprising Met49, Ser55 and ile68, and a light chain variable region sequence (SEQ ID NO: 11) comprising Phe36, Va!44, Phe49 and Ile85 [e.g., antibody 10738] ; or
(ii) a heavy chain variable region sequence (SEQ ID NO: 10) comprising Asn44, Ser55 and Thr93, and a light chain variable region sequence (SEQ ID NO: 11) comprising Phe36, Phe49 and Leu73 [e.g., antibody 10810] .
The anti-HER3 antibody (e) may also be an antibody corresponding to antibody 10738 or 10810, but comprising at Ieast one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4, e.g. substitution in one, two, three or four of such "Xaa" residues.
Examples of a preferred anti-HER3 antibody (f) are antibodies comprising :
(i) a heavy chain variable region sequence (SEQ ID NO: 12) comprising Va!46, Met49, Ser55 and Arg72, and a light chain variable region sequence (SEQ ID NO: 13) comprising Vai21, Val44 and Phe87, and optionally Thr29 [e.g., antibody 11006] ; or
(ii) a heavy chain variable region sequence (SEQ ID NO: 12.) comprising Phe41, Va!46, et49, Ser55 and Arg72, and a light chain variable region sequence (SEQ ID NO: 13) comprising Val21, Val44, Tyr71, Phe87 and Leu 104 [e.g., antibody 11052] . The anti-HER3 antibody (f) may also be an antibody corresponding to antibody 11006 or 1.1052, but comprising at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4, e.g. substitution in one, two, three or four of such "Xaa" residues. it is wel!-known in the art that antibodies exist as different isotypes, such as the human isotypes IgGl, IgG2, IgG3, IgG4, IgAl and igA2, or the murine isotypes IgGl, igG2a, igG2b, IgG3 and IgA, An antibody of the invention may be of any isotype, including IgG, IgM, IgE, IgA, or igD.
Humanized Antibody Compositions
A further aspect of the invention relates to a recombinant antibody composition (or mixture) comprising at least two humanized antibodies of the invention directed against at least two different receptors selected from EGFR, HER2 and HER3. The terms "polyclonal antibody" or "mixture of [monoclonal] antibodies" refer to a composition of two or more different antibody molecules which are capable of binding to or reacting with different specific antigenic determinants on the same or on different antigens, in the context of the present invention, the individual antibodies of a mixture of antibodies bind to different antigenic determinants of at least two HER family receptors. In the case of antibody mixtures containing two different antibodies that bind to the same receptor, the individual antibodies preferably bind to different epitopes of that receptor, more preferably distinct and substantially non-overlapping epitopes.
The terms "pan- HER" or "pan-HER antibody composition" refer to a composition of antibody molecules which are capable of binding to at least two different antigens on at least two HER family receptors. In the context of the present invention, the individual antibodies of an antibody composition bind to different antigenic determinants of the HER family. The individual antibodies of the antibody composition may thus bind to EGFR and HER2, EGFR and HER3, HER2 and HER3, or EGFR, HER2 and HER3, preferably to the three receptors EGFR, HER2 and HER3.
The term "epitope" is used to describe a part of a larger molecule (e.g. antigen or antigenic site) having antigenic or immunogenic activity in an animal. An epitope having immunogenic activity is a portion of a larger molecule that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a larger molecule to which an antibody immunospecificaliy binds as determined by any method known in the art. Antigenic epitopes are not necessarily immunogenic. An antigen is a substance to which an antibody or antibody fragment immunospecificaliy binds, e.g. a toxin, virus, bacteria, protein or DNA. An antigen or antigenic site often has more than one epitope, unless it is very small, and is often capable of stimulating an immune response. Epitopes may be linear or conformational. A linear epitope generally consists of about 6 to 10 adjacent amino acids on a protein molecule that are recognized by an antibody, in contrast, a conformationai epitope consists of amino acids that are not arranged sequentially, but where an antibody recognizes a particular three- dimensional structure. When a protein molecule folds into a three-dimensional structure, the amino acids forming the epitope are juxtaposed, enabling the antibody to recognize the conformationai epitope, in a denatured protein only linear epitopes are recognized. A conformationai epitope, by definition, must be on the outside of the folded protein.
The term "distinct epitopes" refers to the fact that when two different antibodies of the invention bind distinct epitopes, there is less than 100% competition for antigen binding, preferably less than 80% competition for antigen binding, more preferably less than 50% competition for antigen binding, and most preferably as little competition as possible, such as less than about 25% competition for antigen binding. Antibodies capable of competing with each other for binding to the same antigen may bind the same or overlapping epitopes or may have a binding site in dose vicinity of one another, so that competition is mainly caused by steric hindrance. An analysis for "distinct epitopes" of antibody pairs may be performed by methods known in the art, for example by way of binding experiments under saturating antibody conditions using either FACS (fluorescence activated cell sorting) or other flow cytometry analysis on ceils expressing the relevant receptor antigen and individual fluorescent labeled antibodies, or by Surface Plasmon Resonance (SPR) using antigen captured or conjugated to a flow cell surface.
The distinct epitopes are preferably "non-overlapping" in the sense that two different antibodies in a composition of the invention that bind to the same receptor have a sufficiently low competition for antigen binding that the two antibodies are able to bind their respective epitopes simultaneously, it will be understood by persons skilled in the that there can be different degrees of overlap, and that distinct epitopes can be considered to be "non- overlapping" in spite of the presence of some degree of competition, as long as the respective antibodies are able to substantially bind their epitopes. This is generally considered to be the case when the competition for antigen binding between two antibodies is less than about 50%. Methods for determining competition between antibodies are known in the art, for example using Surface Plasmon Resonance (SPR) as described e.g. in WO 2011/1.07957.
Antibodies binding to different epitopes on the same antigen can have varying effects on the activity of the antigen to which they bind, depending on the location of the epitope. An antibody binding to an epitope in an active site of the antigen may block the function of the antigen completely, whereas another antibody binding at a different epitope may have no or little effect on the activity of the antigen alone. Such antibodies may, however, still activate complement and thereby result in the elimination of the antigen-expressing cell, and may result in synergistic growth inhibitory effects when combined with one or more antibodies binding at different epitopes on the same antigen. In the context of the present invention, the epitope is a portion of the extracellular domain of EGFR, HER2 or HERS (either wild-type or mutated) . An anti-EGFR antibody of the invention will thus bind to the extracellular domain of EGFR, an anti-HER2. antibody of the invention will bind to the extracellular domain of HER2, and an anti-HER3 antibody of the invention will bind to the extracellular domain of HERS.
Particular embodiments of this aspect of the invention include, with reference to humanized antibodies (a)-(f) outlined above, compositions comprising : anti-EGFR antibody (a) and anti ■HER2 antibody (c);
anti-EGFR antibody (a) and anti■HER2 antibody (d);
anti-EGFR antibody (a) and ant! HERS antibody (e);
anti-EGFR antibody (a) and anti HERS antibody (f);
anti-EGFR antibody (b) and ant ■HER2 antibody (c);
anti-EGFR antibody (b) and anti■HER2 antibody (d);
anti-EGFR antibody (b) and ant ■HERS antibody (e);
anti-EGFR antibody (b) and ant HERS antibody (f);
anti-HER2 antibody (c) and ant; HERS antibody (e);
anti-HER2 antibody (c) and anti H RS antibody (f);
anti-HER2 antibody (d) and ant -HERS antibody (e); or
anti-HER2 antibodv (d) and anti -HER3 antibody (f).
In one embodiment, the invention relates to a recombinant antibody composition comprising at least one humanized anti-EGFR antibody, at least one humanized anti-HER2 antibody, and at least one humanized anti-HERS antibody.
In some embodiments, the invention relates to an antibody composition comprising at least one humanized anti-EGFR antibody, at least one humanized anti-HER2 antibody, and at least one humanized anti-HERS antibody, wherein:
the at least one humanized anti-EGFR antibody is selected from (a) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: l and the light chain variable region sequence of SEQ ID NQ: 2 or SEQ ID NO: 3, and (b) an antibody comprising the heavy chain variable region sequence of SEQ ID NO:4 and the light chain variable region sequence of SEQ ID NO: 5;
the at least one humanized anti-HER2 antibody is selected from (c) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 6 and the light chain variable region sequence of SEQ ID NO: 7, and (d) an antibody comprising the heavy chain variable region sequence of SEQ ID NO:8 and the light chain variable region sequence of SEQ ID NO: 9; and
the at least one humanized anti-HERS antibody is selected from (e) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11, and (f) an antibody comprising the heavy chain variable region sequence of SEQ ID NO: 12 and the light chain variable region sequence of SEQ ID NO: 13. in the case of an antibody composition comprising one anti-EGFR antibody, one anti-HER2 antibody and one anti-HER3 antibody, the composition may thus comprise, with reference to humanized antibodies (a)-(f) outlined above:
• anti-EGFR antibody (a), anti-HER2 antibody (c), and anti-HER3 antibody (e) ;
• anti-EGFR antibody (a), anti-HER2 antibody (c), and anti-HER3 antibody (f) ;
» anti-EGFR antibody (a), anti-HER2. antibody (d), and anti-HER3 antibody (e) ;
• anti-EGFR antibody (a), anti-HER2 antibody (d), and anti-HER3 antibody (f) ;
® anti-EGFR antibody (b), anti-HER2 antibody (c), and anti-HER3 antibody (e) ;
• anti-EGFR antibody (b), ants-HER2 antibody (c), and anti-HER3 antibody (f) ;
• anti-EGFR antibody (b), anti-HER2 antibody (d), and anti-HER3 antibody (e) ; or » anti-EGFR antibody (b), anti-HER2 antibody (d), and anti-HER3 antibody (f) .
Examples of preferred compositions comprising one anti-EGFR antibody, one anti-HER2 antibody and one anti-HER3 antibody are, e.g . :
» antibodies 10292, 10704 and 10738;
» antibodies 10292, 10704 and 10810;
• antibodies 10292, 10704 and 11006;
» antibodies 10292, 10704 and 11052;
• antibodies 10460, 10704 and 10738;
• antibodies 10460, 10704 and 10810;
• antibodies 10460, 10704 and 11006;
» antibodies 10460, 10704 and 11052;
• antibodies 11294, 10704 and 10738;
» antibodies 11294, 10704 and 10810;
• antibodies 11294, 10704 and 11006; and
• antibodies 11294, 10704 and 11052 .
In a still more preferred embodiment, the antibody composition comprises six humanized antibodies, i .e. two humanized antibodies directed against each of the three receptors EGFR, HER2 and HER3, where each pair of antibodies that bind the same receptor bind to distinct and non-overlapping epitopes of that receptor. This may in particular be a composition comprising anti-EGFR antibodies (a) and (b), anti- HER2 antibodies (c) and (d), and anti-HER3 antibodies (e) and (f) . In this case, one, two, three, four, five or all of the six antibodies may be selected from antibodies 10292, 10460, 11294, 10560, 11302, 10704, 11249, 11145, 10738, 10810, 11006 and 11052.
In a particular embodiment, the antibody composition comprises:
(a) anti - EGFR antibody 10292, 10460, or 11294; (b) anti-EGF antibody 10560 or 11302;
(c) anti-H ER2. antibody 10704 or 11.249;
(d) anti-H ER2 antibody 11145 ;
(e) anti-HER3 antibody 1.0738 or 10810; and
(f) anti-HER3 antibody 11006 or 11052.
Alternatively, any one or more of the antibodies (a)-(f) in this embodiment may comprise at least one additional substitution in any of the heavy chain and/or light chain amino acid residues indicated as "Xaa" in Table 4, e.g . substitution in up to five or six of such "Xaa" residues per antibody for one or more of the antibodies in the composition, such as substitution in one, two, three or four of such "Xaa" residues per antibody for one or more of the antibodies in the composition .
In a preferred embodiment, the antibody composition comprises anti-EGFR antibodies 11294 and 11302, anti-HER2 antibodies 11249 and 11145, and anti-H ER3 antibodies 10738 and 11052. The antibody composition may comprise (a) an antibody comprising the heavy chain variable region sequence of SEQ ID NQ : 43 and the light chain variable region sequence of SEQ ID NQ :44; (b) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 47 and the light chain variable region sequence of SEQ ID NQ : 48; (c) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 51 and the light chain variable region sequence of SEQ ID NO : 52; (d) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 53 and the light chain variable region sequence of SEQ ID NO : 54; (e) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 55 and the light chain variable region sequence of SEQ ID NO : 56; and (f) an antibody comprising the heavy chain variable region sequence of SEQ ID NO : 61 and the light chain variable region sequence of SEQ ID NO : 62.
Although it is possible for the individual antibodies of an antibody mixture of the invention to include antibodies of more than one isotype, they may ail be of the same isotype.
Properties of the Humanized Antibodies and Antibody Compositions
The humanized antibodies of the invention bind to the H ER- or EGFR-famiiy members, EGFR, HER2, or H ER3. The term "HER" stands for "Human Epidermal growth factor Receptor" and is often used interchangeably with the term "ErbB" to characterize the subgroup of the receptor tyrosine kinases (RTKs) consisting of the four members EGFR/ErbB, H ER2/ErbB2, H ER3/ErbB3 and H ER4/ErbB4. Together, these four receptors constitute the "HER family" (or ErbB or EGFR family) receptors.
Binding of one or more antibodies of the invention, in particular a pan-HER antibody composition of the invention, to H ER family receptors preferably inhibits the growth and proliferation of cells expressing the receptors (i .e. typically tumor cells) . The mechanism (s) involved may, for example, include one or more of the following: preventing receptor dimerization, preventing ligand binding, promoting internalization and degradation of the receptor, reducing tyrosine kinase domain (TKD) phosphorylation, reducing receptor signaling, and inducing phagocytosis, CDC and/or ADCC.
As used herein, the term "inhibits growth" (e.g., referring to ceils) is intended to include any measurable decrease in the proliferation (increase in number of cells) or metabolism of a cell when contacted with an anti-HER family antibody or pan-HER antibody composition as compared to the growth of the same cells in the absence of the antibody or composition, e.g. inhibition of growth of a ceil culture by at least about 10%, and preferably more, such as at least about 20% or 30%, more preferably at least about 40% or 50%, such as at least about 60%, 70%, 80%, 90%, 95% or 99%, or even about 100%. Growth inhibition can e.g. be determined in relevant cancer cell lines as described in the examples below,
Bispecific Binding Molecules
In a further aspect, the binding specificities of any two individual antibodies disclosed herein may be combined in one bispecific binding molecule. Such a bispecific binding molecule may have the binding specificities of two antibodies targeting two different receptors selected from EGFR, HER2 and HER3, or it may have the binding specificities of two antibodies targeting the same receptor. For example, a bispecific binding molecule may have the binding specificities of anti-EGFR antibodies (a) and (b), the binding specificities of anti-HER2 antibodies (c) and (d), or the binding specificities of anti-HER3 antibodies (e) and (f). More particularly, a bispecific binding molecule may e.g. have the binding specificities of (1) anti-EGFR antibody 10292, 10460, or 11294, and anti-EGFR antibody 10560 or 11302; (2) anti-HER2 antibody 10704 or 11249, and anti-HER2 antibody 11145; or (3) anti-HER3 antibody 10738 or 10810, and anti- HER3 antibody 11006 or 11052. The bispecific binding molecule may be a dual variable domain antibody, i.e. wherein the two arms of the antibody comprise two different variable domains, or may be in the form of an antibody fragment such as a bispecific Fab fragment or a bispecific scFv.
Nucieic Acid Molecules, Vector, and Production of Antibodies and Antibody Compositions of the Invention
Further aspects of the invention relate to nucieic acid molecules comprising a nucleotide sequence that encodes an antibody of the invention, in particular an antibody selected from the group consisting of antibodies 10292, 10460, 11294, 10560, 11302, 10704, 11249, 11145, 10738, 10810, 11006 and 11052, or encoding a heavy and/or light chain variable region sequence of such an antibody, as well as an expression vectors comprising such a nucleic acid molecule, and host cells comprising the nucleic acid molecule or expression vector, wherein said host cells are capable of expressing an antibody encoded by the nucleic acid molecule. The term "vector" refers to a nucleic acid molecule into which a nucleic acid sequence can be inserted for transport between different genetic environments and/or for expression in a host cell A vector that carries regulatory elements for transcription of the nucleic acid sequence (at least a suitable promoter) is referred to as an "an expression vector". The terms "piasmid" and "vector" may be used interchangeably. Expression vectors used in the context of the present invention may be of any suitable type known in the art, e.g. a piasmid or a viral vector.
An additional aspect of the invention relates to methods for producing humanized recombinant antibodies and compositions comprising the antibodies of the invention. One embodiment of this aspect of the invention relates to a method for producing an antibody as defined herein, comprising providing a host cell capable of expressing the antibody, cultivating said host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody.
In a further embodiment, the invention relates to a method for producing a recombinant antibody composition comprising at least one humanized recombinant anti-EGFR antibody, at least one humanized recombinant anti-HER2 antibody and at least one humanized
recombinant anti-HER3 antibody, the method comprising :
providing at least first, second and third host ceils, wherein the first host ceil is capable of expressing a recombinant anti-EGFR antibody of the invention, the second host ceil is capable of expressing a recombinant anti-HER2 antibody of the invention, and the third host cell is capable of expressing a recombinant anti-HER3 antibody of the invention,
cultivating the first, second and third host cells under conditions suitable for expression of the anti-EGFR antibody, the anti-HER2 antibody and the anti-HER3 antibody, and
isolating the resulting antibodies.
An antibody or antibody composition of the present invention may be produced by methods generally known in the art for production of recombinant monoclonal or polyclonal antibodies. Thus, in the case of production of a single antibody of the invention, any method known in the art for production of recombinant monoclonal antibodies may be used. For production of an antibody composition of the invention comprising a mixture of antibodies, the individual antibodies may be produced separately, i.e. each antibody being produced in a separate bioreactor, or the individual antibodies may be produced together in single bioreactor. If the antibody composition is produced in more than one bioreactor, the purified antibody composition can be obtained by pooling the antibodies obtained from individually purified supernatants from each bioreactor. Various approaches for production of a polyclonal antibody composition in multiple bioreactors, where the cell lines or antibody preparations are combined at a later point upstream or prior to or during downstream processing, are described in WO 2009/129814 (incorporated by reference). in the case of production individual antibodies in a single bioreactor, this may be performed e.g. as described In WO 2004/061.104 or WO 2008/1451.33 (both of which are incorporated herein by reference). The method described in WO 2004/061104 is based on site-specific integration of the antibody coding sequence into the genome of the individual host ceils, while the method of WO 2008/145133 involves an alternative approach using random integration to produce antibodies in a single bioreactor.
Further information regarding methods suitable for preparing the antibodies and compositions of the invention may be found in WO 2012/059857 (incorporated by reference).
Therapeutic compositions
Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient an antibody or antibody composition of the invention. Such compositions are intended for amelioration, prevention and/or treatment of cancer. The pharmaceutical composition may be administered to a human or to a domestic animal or pet, but will typically be administered to humans.
The ratio between the individual antibodies in a therapeutic composition of the invention, or, in the case of individual antibodies of the invention being administered simultaneously, sequentially or separately, will often be such that the antibodies are administered in equal amounts, but this need not necessarily be the case. Thus, a composition of the invention comprising two anti-EGFR family antibodies will often contain them in approximately a 1 : 1 ratio, and a composition comprising three anti-EGFR family antibodies will often contain them in approximately a 1 : 1 : 1 ratio. Similarly, an antibody composition comprising six antibodies, two against each of the receptors EGFR, HER2 and HER3, will often contain them in approximately a 1 : 1 : 1 : 1 : 1 : 1 ratio. Depending on the characteristics of the individual antibodies, however, it may be desirable to use non-equal amounts of the different antibodies. Suitable ratios for the different anti-HER antibodies in compositions of the invention may be determined as described in WO 2010/040356 (incorporated herein by reference), which describes methods for identifying and selecting the optimal stoichiometric ratio between chemical entities in a combinatorial drug product, e.g. a polyclonal antibody composition, to obtain a combinatorial drug with optimal potency and efficacy.
In addition to the humanized recombinant antibodies of the invention or binding fragments thereof, the pharmaceutical composition will further comprise at least one pharmaceutically acceptable diluent, carrier or excipient. These may for example include preservatives, stabilizers, surfactants/wetting agents, emulsifying agents, solubllizers, salts for regulating the osmotic pressure and/or buffers. Solutions or suspensions may further comprise viscosity- increasing substances, such as sodium carboxymethylceilulose, carboxymethylceiiulose, dextran, polyvinylpyrrolidone or gelatin. A suitable pH value for the pharmaceutical composition will generally be in the range of about 5.5 to 8.5., such as about 6 to 8, e.g. about 7, maintained where appropriate by use of a buffer. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer to e.g. cancer patients by conventional administration routes known in the art. Similarly, the pharmaceutical compositions of the invention may be prepared in a manner known per se for preparation of recombinant antibody compositions. For further information on formulation, administration, etc., see PCT/IB2011/054834.
Therapeutic uses of antibodies and compositions of the invention
The antibodies and compositions of the present invention may be used for the treatment or amelioration of a disease in a mammal, in particular treatment of cancer in humans. The term "treatment" as used herein refers to administration of an antibody or, preferably, antibody composition of the invention in a sufficient amount to ease, reduce, ameliorate or eradicate (cure) symptoms or disease states. Administration of two or more pan-HER antibodies of the invention will generally be by way of simultaneous administration of the antibodies, preferably in the form of a composition containing all of the pan- HER antibodies to be used for treatment. However, it is also possible to administer two or more antibodies of the invention separately. References herein to e.g. administration of a recombinant antibody composition comprising at least two anti-HER family antibodies should therefore be understood as encompassing not only administration of a composition comprising the at least two antibodies as such, but also separate administration of the antibodies. Combinations of two or more antibodies of the invention can thus be administered simultaneously, sequentially or separately. One embodiment of the invention is a method of preventing, treating or ameliorating one or more symptoms associated with cancer in a human or other mammal, comprising administering an effective amount of the pharmaceutical antibody composition of the present invention to said mammal. A particular embodiment relates to a method for treating a patient, typically a human patient, with a disorder characterized by expression or overexpression of or dependency on any one or more of the EGFR family receptors EGFR, HER2 and HER3, in particular cancer, the method comprising administering to said patient a recombinant antibody composition or
pharmaceutical composition as defined herein. The term "HER dependency" refers to a cancer cell with dependency on one or more of the HER family receptors for maintaining malignant properties such as proliferation, growth, motility, invasion, survival and/or chemo resistance. Dependency may be caused by receptor overexpression, receptor mutations, autocrine growth factor production, and/or cross-talk with other receptor systems. in a further embodiment, the invention relates to a method for treating cancer in a patient, typically a human patient, having acquired resistance to treatment with an antibody and/or a tyrosine kinase inhibitor (TKi), the method comprising administering to said patient an effective amount of a recombinant antibody composition or pharmaceutical composition as defined herein.
Based upon a number of factors, the following tumor types in particular may be indicated for treatment with an antibody composition of the invention: breast, ovarian, gastric, colon, rectum, prostate, bladder, pancreas, melanoma, head and neck, and non-small cell lung cancer. Antibody compositions of the invention are contemplated to be particularly applicable to treatment of cancers that overexpress EGFR or HER2, for example certain epithelial cancers such as many breast cancers, ovarian cancers and gastric (stomach) cancers.
In one embodiment, antibody compositions of the invention are used to treat a patient with pancreatic cancer. The patient may have a KRAS mutation.
In one embodiment, the patient has not been treated for cancer previously, in another embodiment, the patient has been treated for cancer previously. The patient may have been treated with cetuximab, trastuzumab, or pertuzumab previously. The cancer in the patient may have acquired resistance to cetuximab, trastuzumab, or pertuzumab.
In connection with each of these indications, two main clinical pathways are contemplated, namely I) adjunctive therapy in connection with at least one additional therapeutic treatment or 2) as a monotherapy.
1) Adjunctive therapy: In adjunctive therapy, also known as combination therapy, patients will be treated with antibodies of the present invention in combination with at least one additional therapeutic treatment, typically a chemotherapeutic or antineoplastic agent and/or radiation therapy. Alternatively or additionally, the composition of the invention may also be used in combination with a different anti-cancer antibody, e.g. an antibody targeting VEGF. The primary cancer targets listed above may thus be treated by administration of an antibody or composition of the invention in addition to standard first line and second line therapy. Protocol designs will address effectiveness as assessed e.g. by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. Such dosage reductions may allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent.
By combining the antibody compositions of the invention with agents known to induce terminal differentiation of cancer cells, the effect may be improved further. Such compounds may, for example, be selected from the group consisting of retinoic acid, trans-retinoic acids, cis- retinoic acids, phenyibutyrate, nerve growth factor, dimethyl sulfoxide, active form vitamin D3, peroxisome pro! ifera tor-activated receptor gamma, 12-O-tetradecanoylphorbol 13- acetate, hexamethylene-bis-acetamide, transforming growth factor-beta, butyric acid, cyclic AMP, and vesnarinone. Preferably, the compound is selected from the group consisting of retinoic acid, phertyibutyrate, a!i-trans-retinoic acid and active form vitamin D.
Pharmaceutical articles comprising an antibody composition of the invention and at least one chemotherapeutic or antineoplastic compound may be used as a combination treatment for the simultaneous, separate or successive administration in cancer therapy. The
chemotherapeutic compound may by any chemotherapeutic agent suitable for treatment of the particular cancer in question, for example an agent selected from the group consisting of alkylating agents, for example platinum derivatives such as cispiatin, carbopiatin and/or oxaliplatin; plant aikoids, for example pac!itaxe!, docetaxei and/or irinotecan; antitumor antibiotics, for example doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and/or mitomycin; ;
topoisomerase inhibitors such as topotecan; and/or antimetabolites, for example fiuorouraci! and/or other fluoropyrimidines.
It is also contemplated that antibody composition of the invention may be used in adjunctive therapy in connection with tyrosine kinase inhibitors. These are synthetic, mainly quinazoline- derived, low molecular weight molecules that interact with the intracellular tyrosine kinase domain of receptors and inhibiting ligand-induced receptor phosphorylation by competing for the intracellular Mg-ATP binding site. Several tyrosine kinase inhibitors that block HER2 kinase are currently in clinical development. Some of these also target EGFR or other EGFR family receptors. For a review of these TKIs see Spector et al. (2007) Breast Cancer Res. 9(2) : 205. Pharmaceutical articles comprising an antibody composition of the invention and at least one Ki targeting HER2 may thus also be used as a combination treatment for the simultaneous, separate or successive administration in cancer therapy.
In other embodiments, the antibody compositions of the present invention may be used in combination with other antibody therapeutics, e.g. an antibody against VEGF (e.g. Avastin®). In yet other embodiments, the antibody compositions of the present invention may be used in combination with an agent known to stimulate cells of the immune system, such combination treatment leading to enhanced immune-mediated enhancement of the efficacy of the antibody compositions of the invention. Examples of such immune-stimulating agents include recombinant inter!eukins (e.g. IL-21 and IL-2).
2) Monotherapy: In connection with the use of the antibody composition in accordance with the present invention in monotherapy of tumors, the antibody composition may be administered to patients without concurrent use of a chemotherapeutic or antineoplastic agent, i.e. as a stand-alone therapy.
Immunoconjugates
Another option for therapeutic use of the compositions of the invention is in the form of immunoconjugates, i.e. antibodies conjugated to one or more anti-cancer agents, in particular in the case of compositions of the invention that bind distinct epitopes, it is contemplated that this may generate a cross-linked antibody-receptor lattice on the ceil surface, thereby potentially resulting in an increased level of receptor internalization as compared to the use of a single monoclonal antibody. Conjugation of one or more of the individual antibodies of such a composition to one or more anti-cancer agents therefore has the potential to specifically and effectively deliver the conjugated anti-cancer agents to the interior of tumor ceils, thereby augmenting the effect of the antibody composition of the invention to provide an improved tumor cell-killing activity.
Various types of anti-cancer agents may be conjugated to the antibodies of the invention, including cytotoxic agents (including conventional chemotherapy agents and other small molecule anti-cancer drugs), cytokines (in which case the conjugate may be termed an "immunocytokine"), toxins (in which case the conjugate may be termed an "immunotoxin") and radionuclides, and a few immunoconjugates have already been approved for clinical use. These include Zevalin® (a murine anti-CD20 antibody conjugated to ¾0Y), Bexxar® (a murine anti-CD20 antibody conjugated to 131I) and My!otarg® (a humanized anti-CD33 antibody conjugated to calicheamicin). Other immunoconjugates that have been tested in clinical trials include antibodies conjugated to e.g. doxorubicin or a maytansinoid compound, immunotoxins that have been tested in clinical trials include several antibodies conjugated to a truncated Pseudomonas exotoxin A. An immunocytokine comprising a humanized EpCA antibody conjugated to IL-2 has also been tested.
In the case of antibodies of the invention conjugated to cytotoxic agents, these may e.g. belong to any of the major classes of chemotherapy drugs, including alkylating agents (e.g. carboplatin, cisplatin, oxaiiplatin), antimetabolites (e.g. methotrexate, capecitabine, gemcitabine), anthracyclines (e.g. bleomycin, doxorubicin, mitornycin-C) and plant alkaloids (e.g. taxanes such as docetaxel and paciitaxei, and vinca alkaloids such as vinblastine, vincristine and vinorelbine). Since the use of immunoconjugates specifically directs the anticancer agent to the tumors, and in particular to the interior of the tumor cells subsequent to internalization, immunoconjugates based on the antibodies of the invention may
advantageously be based on highly cytotoxic agents such as calicheamicin or maytansine derivatives, or on toxins such as bacterial toxins (e.g. Pseudomonas exotoxin A, diphtheria toxin) or plant toxins (e.g. ricin). The conjugated anti-cancer agent in an immunoconjugate is generally linked to the antibody by means of a labile linker that is relatively stable in serum but which allows release of the agent when the immunoconjugate is internalized into the target cell. Suitable linkers include, for example, chemical linkers that are stable at neutral pH in serum but are subjected to acid hydrolysis in the mildly acidic conditions within the lysosomes subsequent to internalization, disulfide linkers that are cleaved by intracellular thiols, and peptide linkers that are stable in serum but which are subjected to enzymatic cleavage in intracellular compartments.
Various conjugation arrangements can be envisioned in compositions containing two or more antibodies of the invention. For example, with two antibodies it would be possible to conjugate the antibodies to two or more different anti-cancer drugs or to conjugate one antibody to a prodrug which is activated by an agent such as an enzyme conjugated to the other antibody. The general concept of antibody-directed enzyme prodrug therapy (ADEPT) has been described for monoclonal antibodies, where a prodrug is activated by an enzyme targeted to the tumor by a mAB-enzyme conjugate, but the present invention may provide an opportunity for tailoring this approach to particular conditions, it may thus be possible to specifically increase tumor cell killing while sparing or reducing damage to normal tissues.
For further information on anti-cancer immunoconjugates, see Wu et ai. (2005) Nature Biotechnology 23(9) : 1137-1146; Scbrama et al. (2006) Nature Reviews/Drug Discovery 5: 147-159; and Rohrer (2009) chimica oggi/Chemistry Today 27(5) : 56-60.
Compositions of the invention comprising antibodies directed against two or more EGFR family receptors may contain a single antibody in the form of an immunoconjugate, or they may contain two or more antibodies in the form of an immunoconjugate, e.g. one or possibly two immunoconjugates targeting each of the receptors EGFR, HER2 and HER3.
Dose and Route of Administration
The antibody compositions of the invention will be administered in an effective amount for treatment of the condition in question, i.e. at dosages and for periods of time necessary to achieve a desired result. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the antibodies are being administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments.
An effective amount for tumor therapy may be measured by its ability to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression, e.g. by reducing tumor size. The ability of an antibody or composition of the invention to inhibit cancer may be evaluated by in vitro assays, e.g. as described in the examples, as well as in suitable animal models that are predictive of the efficacy in human tumors. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.
While specific dosing for antibodies in accordance with the invention has not yet been determined, certain dosing considerations can be determined through comparison with a similar product (e.g. a monoclonal antibody directed against HER2 or EGFR) that has been approved for therapeutic use. It is thus contemplated that an appropriate dosage of an antibody composition of the invention will be similar to the recommended dosage for the anti- HER2 monoclonal antibody trastuzumab (Herceptin®) or the anti-EGFR monoclonal antibody panitumumab (Vectibix®). Depending on the particular condition, Herceptin® is administered (by way of infusion) for treatment of breast cancer at either an initial dose of 4 mg/kg and subsequent weekly doses of 2 mg/kg, or an initial dose of 8 mg/kg and subsequent doses of 6 mg/kg every three weeks, while Vectibix® is administered at a dose of 6 mg/kg every 14 days.
It is contemplated that a suitable dose of an antibody composition of the invention will be in the range of 0.1-100 mg/kg, such as about 0.5-50 mg/kg, e.g. about 1-20 mg/kg. The antibody composition may for example be administered in a dosage of at least 0.25 mg/kg, e.g. at least 0.5 mg/kg, such as at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2 mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg, e.g. at least 5 mg/kg; and e.g. up to at most 50 mg/kg, such as up to at the most 30 mg/kg, e.g. up to at the most 20 mg/kg, such as up to at the most 15 mg/kg. Administration will normally be repeated at suitable intervals, e.g. once every week, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the responsible doctor, who may optionally increase or decrease the dosage as necessary.
Three distinct delivery approaches are contemplated for delivery of the antibodies of the invention. Conventional intravenous deliver will presumably be the standard delivery technique for the majority of tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favourable for obtaining high dose of antibody at the tumor and to minimize antibody clearance. Similarly, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion may allow the obtainment of a high dose of the antibody at the site of a tumor and minimise short term clearance of the antibody.
As with any protein or antibody infusion-based therapeutic product, safety concerns are related primarily to (i) cytokine release syndrome, i.e. hypotension, fever, shaking, chills, (ii) the development of an immunogenic response to the protein (i.e. development of human antibodies by the patient to the recombinant antibody product), and (Hi) toxicity to norma! cells that express the HER family receptors, e.g. many epithelial cells. Standard tests and fo!iow-up procedures are utilised to monitor any such safety concerns.
Diagnostic Uses and Compositions
The antibodies of the present invention also are useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies can be used to detect and/or measure the level of EGFR, HER2, or HER3 in a sample from a patient (e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, serum, bowel fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELiSA), chemiluminescence assays,
radioimmunoassay, and immunohisto!ogy. The invention further encompasses kits (e.g., diagnostic kits) comprising the antibodies described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. AH publications and other references mentioned herein are incorporated by reference in their entirety, in case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and embodiments, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The materials, methods, and examples are illustrative only and not intended to be limiting.
The following examples are meant to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described conditions and parameters normally encountered in the art which are obvious to those skilled in the art are within the spirit and scope of the present invention. The terms "antigen-binding fragment" and "antigen- binding portion" are used interchangeably herein. EXAMPLES
Example 1: Hisma ss^atson of c smersc antsbodses
Iderstsfscatson of acceptor frameworks and critical positions for mutatsor*
The method chosen for the humanization was based on complementarity determining region (CDR) grafting followed by back mutation of critical residues using a combinatorial library approach, where all combinations of up to 13 back mutations were evaluated simultaneously.
The CDRs of the donor murine antibodies were grafted into the closest human V-region acceptor framework, which was found by comparing the V region amino acid sequences of the donor antibodies with the human germline repertoire of V and J region sequences (i GT reference directory). The closest germline V and J regions identified for each antibody are shown in Table 5 below.
For 1277VL, the closest human germline V-region was IGKV2-30*02. However, since the IGKV2 family is rarely used in the human repertoire, a second acceptor framework was also selected from the IGKV1 family. Each of the two frameworks were used for generation of a VL back mutation library and combined with the single 1277 VH back mutation library.
Since CDR grafting alone may not be sufficient to recreate the binding specificity and affinity, and thus biological activity, of a rodent antibody, back mutations may have to be introduced at critical positions. Potentially critical positions include those that are somatically hypermutated in the donor antibody, positions that may be in direct contact with the antigen or influencing CDR structure (structure determining residues or Vernier zone residues), positions in the VH/VL interface or responsible for the VH/VL packing angle, and positions that are occupied by statistically rare (as compared to the antibody repertoire) or structurally unfavorable residues. These positions can be identified using information available in the literature and in antibody databases (e.g., Padian (1994) Moi. Immunol. 31 : 169-217; Honegger and Piuckthun (2001) j.Mol.Biol. 309: 657-670; http://www.bioc.uzh.ch/antibody; Martin and Thornton (1996) j.Mol.Biol.. 263: 800-815; http://www.bioinf.org.uk/abs/; Foote and Winter (1992.) J.Mol.Biol. 2.24: 487-499), or by performing structural modeling of the in silico grafted sequence. A combination of these two approaches was used to identify potentially critical positions for back mutation in each of the antibodies.
In addition to the back mutation positions, exposed unwanted sequence motifs in the CDRs were also identified. These motifs included sites for asparagine deamidation (Asn-Giy), aspartate isomerization (Asp-Gly) and methionine oxidation. The identified sequence motifs were altered by conservative substitution or replacement with a frequently occurring amino acid residue at one of the positions (as opposed to back mutation to the murine sequence). A maximum of 13 critical positions were identified and inciuded in the iibrary design for each antibody (Table 5) . The number of positions was seiected on the basis of the size of the resulting back mutation libraries. For example, if 13 positions are varied between two different amino acids (e.g . human or murine residue) this yields 8192 variants when combined into one molecular Iibrary. The location of the identified positions in each antibody is shown in the appended sequence listing, where amino acid residues indicated by "Xaa" are potentially critical positions selected for mutation.
Table 5; Design of libraries for htsmanizatiors
Figure imgf000061_0001
*Number of positions where back mutations were introduced or unwanted sequence motifs altered .
Generation of back mutation libraries
Back mutation libraries for each VH and VL sequence were synthesized by PGR gene assembly of overlapping DNA oiigos spanning 60-80 base pairs of the sequence. The light chain constant region was added by overlap extension PGR to generate full-length Iight chain genes.
Molecular libraries of humanized antibody variants were prepared by sub-cloning of the VH and Iight chain libraries for each antibody into a mammalian expression vector followed by transient expression of individual antibody variants in HEK293 cells in 384-weli format as described elsewhere (Meijer et al . (2009) Methods Mol Biol . 525 : 261-77) . Expression supernatants were harvested and used for screening .
Off-rate screening of hymani^ation libraries The library expression supernatants were screened In a sandwich ELISA employing IgG capture by anti-human IgG Fc coated at low density followed by detection with monovalent biotinyiated antigen. This ELJSA setup allowed for sensitive and reliable ranking of binding affinity without interference from avidity effects or varying expression levels of individual clones. In total, 24 384-weli plates were used for each library screening, corresponding to 8832 individual wells and a library sampling of approximately 1 (p=0.65 for retrieving a distinct library member). 5 μΙ of each library expression supernatant was incubated with coated anti-human igG Fc capture antibodies at 4°C overnight to ensure that all supernatants, regardless of antibody expression level, reached equilibrium binding. Next, wells were washed and biotinyiated antigen (human EGFR, HER2 or HER3; Sino Biological, Beijing, P.R. China; biotinyiated in-house) was added at a concentration previously determined to be sufficient for saturation of the chimeric antibody standards. The plates were washed and the antigen was allowed to dissociate from the captured antibodies for a predetermined time interval depending on the measured dissociation of the chimeric parent antibody standard. Finally, streptavidin-peroxidase polymer (Sigma) was added and the plates were developed using TMB-pius substrate (Kem-En-Tec Diagnostics, Taastrup, Denmark).
Approximately 100 hits from each library that yielded an OD signal similar to or higher than that of the chimeric parent antibody were subjected to off-rate ranking using an Octet® QK384 instrument (Fortebio, Menlo Park, CA). Protein G biosensors (Fortebio) were used for capturing of antibody from 40 μΙ of expression supernatant followed by incubation with human or cynomo!gus antigen at 200 n . Human antigens were obtained from Sino Biologicals and cynomolgus antigens were produced in-house by transient expression in CHO or HEK293 cells (Koefoed et al. (2011) mAbs, 3 : 6, 1-12). Subsequently, the biosensors were incubated in PBS and the dissociation of antigen was recorded for 20 min to aliow for a reliable determination of the dissociation rates. The responses were globally fitted to a Langmuir 1 : 1 binding model for calculation of dissociation constants. Overall, multiple hits from each library were found to have dissociation rates from both human and cynomolgus antigen similar to or slower than that of the parent antibody.
Sequence nalys s
Piasmids encoding the hits selected for off-rate ranking were subjected to DMA sequencing
(MWG Biotech, Ebersberg, Germany), and the obtained sequences were aligned and compared to the in silico generated CDR-grafted V regions. Alignments of selected hits are shown in
Figures 1-6. Ail the hits from the screening of the initial libraries based on antibodies 1277 and
1565 were found to have retained the deamidation site (Asn-Giy) in CDRL1 and CDRH2, respectively, thus indicating the importance of the motif for the interaction with the target.
However, only a single replacement mutation (Asn to Ser) was attempted in both cases, and it is quite likely that binding variants devoid of the sequence motif can be generated by saturated mutagenesis of one or both positions that make up the motif. Screening of the libraries generated by PCR-based saturated mutagenesis of the deamidation sites yielded hits devoid of this unwanted sequence motif (Figure 1 and 2) . Potently binding antibody variants devoid of unwanted sequence motifs were found in ail the other libraries.
Between four and ten hits from each library screening were selected on the basis of retained or improved binding to human and cynomoigus antigen, the number of back-mutations and absence of unwanted sequence motifs for expression in larger scale and purification by protein A chromatography. One of the humanized variants, antibody 11006, was found to have a fortuitous mutation in CDRL1 (I29T; SEQ ID NO: 13 and Figure 6) that was not part of the library design, but was nevertheless selected for expression due to improved dissociation rate and removal of an aspartate isomerization site in CDRH2.
Kinetic binding analysis of humanized variants by surface pfasmon resonance
Kinetic binding analysis of the purified humanized variants was performed on a ProteOn™ XPR36 biosensor (BioRad, USA) employing an igG capture assay as described by Canziani et ai . (Anal. Biochem . (2004) 325 : 301-307) that allows for measurement of antibody affinities of whole igG molecules against soluble antigen under monovalent conditions. Briefly,
approximately 5000 resonance units (RU) of a monoclonal mouse anti-human IgG Fc antibody (GE Healthcare, Denmark) was conjugated to a GLC chip surface (BioRad, USA) according to the manufacturer's instructions, followed by capture of individual antibodies of the invention or a negative control on the anti-Fc sensor surface. The densities of captured antibodies were optimized for each clone, so that the binding of the highest antigen concentration employed in the assay did not exceed ~30 RU. Next, 250 μΙ monovalent antigen (Sino Bioiogicals) was injected at a flow rate of 50 μΙ/min in serial threefold dilutions from 100 n stock to generate response curves. The chip surface was regenerated between cycles by stripping the captured antibody/antigen complexes off the surface with a 10-second injection of 3 M MgCI2 (GE Healthcare, Denmark) repeated three times. Finally, binding responses were fitted to a Langmuir 1 : 1 binding model for calculation of the on-rate (kon or ka), off- rate (koff or kd) and affinity (KD) constants using double referencing . The results of the kinetic binding analysis show that the selected variants have retained or even improved affinity for the human and cynomoigus antigen as compared to the chimeric parent antibodies (Table 6) .
Table 6: Binding affinity of chimeric parent antibodies and humanized antibodies
Ab Source library Human antigen Cynomoigus antigen
ID (specificity) ka (M'V1) kd (s"1} K0 (M) ka (M'V1) kd Cs"1}
1277 chimeric 9.4E+05 3.5E-04 3.7E-10 6.6E+05 3.9E-04 5.9E-10 (EGFR)
10292 1277 (EGFR) 1.5E+06 4.8E-04 3.2E-10 7.9E+05 2.7E-04 3.5E-10
10460 1277.A (EGFR) 1.3E+06 5.3E-04 4.1E-10 9.7E+05 5.2E-04 5.3E-10
11294 1277A (EGFR) 3.4E+05 1.8E-04 5.3E-10 3.7E+05 2.1E-04 5.6E-10 1SSS chimeric 1.7E+06 5.8E-04 3.5E-10 5.8E+05 1.6E-02 2.8E-08 (EGFR)
10560 1565 (EGFR) 1.7E+06 4.6E-04 2.7E-10 8.9E+05 2.7E-03 3.1E-09
11302 1565 (EGFR) 4.9E+Q5 9.6E-05 2.0E-10 4.2E+05 4.9E-04 1.2E-09
4384 chimeric 4.0E+05 3.0E-04 7.5E-10 1.8E+05 5.0E-04 2.9E-09 ( ER2)
10704 4384 (HER2) 3.6E+05 1.4E-04 3.9E-10 1.9E+05 2.7E-04 1.4E-09 1249 4384 (HER2) 2.2E+05 1.1E-04 5.0E-10 1.5E+05 3.8E-04 2.5E-09
4S17 chimeric 2.6E+05 2.9E-04 1.1E-09 2.3E+05 8.6E-04 3.7E-09 ( ER 2)
11145 4517 (HER2) 1.27E+05 1.24E- 9.8E-10 5.3E+04 6.3E-04 1.2E-08
04
5038 chimeric 3.0E+05 4.8E-04 1.6E-09 4.6E+05 4.1E-04 8.9E-10 ( ER 3)
10738 5038 (HERS) 2.6E+05 1.9E-04 7.5E-10 5.4E+05 2.9E-04 5.4E-10
10810 5038 (HERS) 1.9E+05 2.QE-Q4 1.1E-09 4.7E+05 3.4E-04 7.1E-10
5082 chimeric 9.1E+05 7.3E-05 8.0E-11 1.7E+06 1.6E-04 9.8E-11 (HER3)
1006 5082 (HER3) 7.4E+05 <2E-6 D* 1.5E+06 8.7E-05 5.9E-11
11052 5082 (HER3) 8.7E+05 1.6E-04 1.8E-10 1.3E+06 2.6E-04 1.9E-10
*KD could not be determined due to a very slow off- rate. Estimated to be in the picomolar range.
Irs vitro functional evaluation of humanized variants
Humanized antibody variants were tested for functional effect in a viability assay in combination with a chimeric "partner antibody" in an antibody mixture containing two antibodies against different epitopes of a particular target (where "partner antibody" refers to the fact that antibodyl277 variants (anti-EGFR) were tested together with the chimeric anti- EGFR antibody 1565, antibody 4384 variants (anti- HER2) were tested in combination with the chimeric anti-HER2 antibody 4517, and so forth) to determine if the functional synergy between the two antibodies targeting the same receptor was preserved after humanization. Each humanized variant was tested in two cell lines and compared to the parental mixture of two chimeric antibodies and to a negative control antibody. The ceil lines used were selected on the basis of their previously determined receptor-dependency, i .e., A431 NS epidermoid, H358 non-small cell lung , and FaDu head and neck cancers for EGFR, OE19 esophageal and BT474 breast cancer for HER2, and MDA-MB-175 VII and MCF-7 breast cancer for HER3. In addition, a combination of six humanized variants (11.294, 11302, 11249, 11145, 10738 and 11052; Humanized Pan-HER) was tested in a number of cell lines and compared to the combination of the six chimeric antibodies ( 1277, 1565, 4384, 4517, 5038 and 5082; Chimeric Pan-HER) . The cell lines used, N87 gastric, FaDu head and neck, A431NS epidermoid, OE19 esophageal, HN5 head and neck, MDA-MB-175 VII breast and FE-280 endometrial cancer, were selected on the basis of their previously determined dependency on the HER family receptors. Prior to performing the viabiiity assay the appropriate antibodies and antibody mixtures were diluted to a final total antibody concentration of 100 g/ml in appropriate media supplemented with 0.5-2% FBS and 1% P/S (penicillin/streptomycin), yielding a final total antibody concentration of 50 pg/mi in the well containing the highest antibody concentration. A threefold serial dilution of the antibodies was then performed in a 384-weli plate, followed by addition of relevant numbers of ceils to the experimental wells. The CF-7 cells were also stimulated with 1 n heregulin beta. The plates were incubated for 4 days in a humidified incubator at 37°C. WST-1 reagent (Roche Applied Science, Mannheim, Germany) was added to the plates and the plates were incubated for 1-3 h at 37°C. Plates were transferred to an orbital plate shaker for one h and the absorbance was measured at 450 and 620 nm
(reference wavelength) using an ELISA reader. The percentage of metabolicaily active cells (MAC) is calculated as a :
Figure imgf000065_0001
it. - ODmedium )
The in vitro activity of selected humanized antibody variants is shown in Figures 7-15 and 17- 19. The results show that ail of the selected humanized variants display an anti-pro!iferative effect when combined with their chimeric or humanized partner that is very similar to the effect of the relevant mixture of the two chimeric parent antibodies. Furthermore, the combination of six humanized variants also displays an effect very similar to the effect of the combination of the six chimeric parent antibodies (Figure 20).
Specificity of umanize variants (cross-reactivity)
The chimeric parent antibodies and selected humanized variants were tested for binding to EGFR, HER2 and HER3 from humans, cynomolgus monkeys and mice, as we!I as human and murine HER4, to determine whether the humanization had introduced any changes in the cross- eactivity pattern.
Antibody-antigen binding was measured by ELISA with coated antigens. Human antigens were obtained from Sino Biologicais. AH other antigens were produced in-house by transient expression in CHO or HEK293 ceils. Chimeric and humanized antibodies, as well as an isotype control antibody, were incubated with the coated antigens at different concentrations. After wash, bound antibodies were detected by HRP- (horse radish peroxidase)-conjugated secondary antibodies. The OD signal from 40 nM antibody, measured at 450 nm using an ELISA reader, was scored from negative (-; OD<0.1) to strongly positive (+ + + ; OD>2.5).
The results, shown in the table in Figure 16, demonstrate that cross-reactivity between the respective human and cynomolgus antigens is conserved in all humanized antibody variants, and that no new reactivity to members of the epidermal growth factor receptor family has been introduced.
Summary and conclusions
A number of humanized variants of the chimeric anti-EGFR, anti-HER2 and anti-HER3 antibodies disclosed in PCT/IB2011/054834 were produced by screening of CDR-grafted libraries generated by back mutation of potentially critical framework positions and in some cases by alteration of unwanted CDR sequence motifs. Approximately 100 hits from each library selected for binding affinity to the relevant target antigen were subjected to off-rate ranking, and variants with a dissociation rate similar to or siower than that of the parent chimeric antibody were selected and sequenced . Between four and ten hits from each library screening were selected on the basis of retained or improved binding to human and cynomolgus antigen, number of back-mutations and absence of unwanted sequence motifs for larger scale expression and purification. Selected purified humanized antibody variants were subjected to a kinetic binding analysis to determine binding affinity to human and cynomolgus antigen, to in vitro functional analysis in a viability assay in combination with a chimeric partner antibody binding to a different epitope of the same receptor, and to a cross-reactivity assay.
Each of the humanized variant antibodies 10292, 10460, 11294, 10560, 11302, 10704, 112449, 11145, 10738, 10810, 11006 and 11052 were found to exhibit functional properties that were very similar to those of the original chimeric parent antibody from which they were derived, including :
® similar or higher binding affinity;
® similar or slower dissociation rate;
® binding to the same human and cynomolgus antigen combined with lack of binding to the mouse antigen or to other EGFR family receptors; and
® highly similar anti-proliferative effects in two different cell lines when tested in a
functional in vitro assay in combination with the chimeric partner antibody.
These results thus demonstrate that the humanized antibody variants of the invention have functional characteristics that are highly similar to the respective parent chimeric antibodies from which they are derived. This strongly suggests that mixtures of the humanized antibodies of the invention, e.g. mixtures containing one or two such antibodies against each of the EGFR family receptors EGFR, HER2 and HER3, can be expected to demonstrate anti-cancer effects in vivo that are similar to the effects of the mixtures of the parent chimeric antibodies described in PCT/IB2011/054834. EXAMPLE 2; Two morsociorsai antsbodses against rsors-oveHappi g epitopes ors EGFR, HER2 or HERS display synergistic ire vitro growth inhibitory activity and effectively induce target do rs-reguiation
Antibodies against non-over!apping epitopes on EGFR (i.e., 1277 and 1565), HER2 (i.e., 4384 and 4517), and HER3 (i.e., 5038 and 5082) as illustrated in Figure 21A, were tested for their ability to inhibit the growth and proliferation of the cancer ceil lines A431NS, HCC2.02, and M DA- MB- 175- VI I, respectively, using a viability assay. Antibody treatments consisted of antibodies to each receptor administered either alone or in the following combinations: 1277 and 1565 mixture, 4384 and 4517 mixture, and 5038 and 5082 mixture. Cellular damage will inevitably result in loss of the ability of the ceil to maintain and provide energy for metabolic cell function and growth. Metabolic activity assays are based on this premise and usually measure mitochondrial activity. The Cell Proliferation Reagent WST-1 (Roche Cat. No 11 644 807 001) is a ready-to-use substrate that measures the metabolic activity of viable cells, it is assumed that the metabolic activity correlates with the number of viable cells. In this example, the WST-1 assay was used to measure the number of metaboiical!y active ceils after treatment of cancer cells with different concentrations of antibodies for 96 hours.
Prior to performing the WST-1 assay, the appropriate antibodies and antibody mixes were diluted to a final total antibody concentration of 100 Mg/ml in appropriate media supplemented with 2% of FBS and 1% P/S yielding a final total antibody concentration of 50 μς/mi in the well containing the highest antibody concentration. A threefold serial dilution of the antibodies was then performed. Relevant numbers of cells were then added to the experimental wells in a 384-well plate. The plates were incubated for 4 days in a humidified incubator at 37°C. WST- 1 reagent was then added to the plates and the plates were incubated for one hour at 37°C. Plates were transferred to an orbital plate shaker for one hour and the absorbance was measured at 450 and 620 nm (reference wavelength) using an ELISA reader. The amount of metaboiica!ly active cells (MAC) is calculated as a percentage of the untreated control as follows:
Figure imgf000067_0001
The in vitro effects of antibody treatment showed that mixtures of antibodies are superior to the individual antibodies to each of the three HER receptors tested (Figure 21B).
Furthermore, analysis of EGFR, HER2. and HER3 levels in ceil iysates isolated from antibody treated A431NS, HCC2.02 and MDA- B-175-VII ceils (20 pg/mi total antibody for each treatment for 48 hours) by Western Blot analysis showed that antibody treated ceils exhibited reduced levels of EGFR, HER2 and HER3 compared to untreated cells (Figure 21C).
This example demonstrates that two antibodies against EGFR, HER2 or HER3 display synergistic in vitro growth inhibitory activity and effectively induce target down-regulation. EXAMPLE 3; Pan-HER is broadiv inhibitory in a large number of ceil tines of different tsssue origm and genetic background
Mixtures of antibodies against non-overlapping epitopes on EGFR, HER2, and/or HER3 were tested for their ability to inhibit the growth and proliferation of a broad range of cancer cell lines. The effects of treatment with Pan-HER (a mixture of six monoclonal antibodies against EGFR, HER2 and HERS; antibodies 1277, 1565, 4384, 4517, 5038, and 5082), antibody mixtures targeting two HER family members (i .e., EGFR and HER2, EGFR and HER3, and HER2 and HER3), and antibody mixtures targeting one HER family member (i .e. , EGFR, HER2 and HER3) were measured in the foliowing cell lines: HNS, MDA-MB175-VII, HCC827, 87, A431NS, FaDu, OE19, SW948, BT474, R G- l, TEH, GEO, H358, CALU-3, H292, HCC202, LS174T, ZR-75-30, H 1975, KYSE520, AU-565, CAPAN- 1, IGR-OV1, OE33, PK-1, CFPAC-1, BxPC3; A431, SW1463, COL0678, H820, COLO680N, ASPCl, HCC1937, H661 , MFE-280, OVCAR-3, OVCAR-5, SK-BR-3, SW403, OVCAR-8, RL95-2, RMUG-S, SW837, T84, CAPAN-2, GP5d, CaC02, BT2Q, MDA-MB-468, DU145, A549, CAL-120, EBC1, H 1993, H226, HEC-108, LoVo, Panc08.13, RT-112, U2-OS, DLD-1, SKOV3, H460, KATOIII, MDA-MB-134-VI, MKN-45, PANC-i, RT-4, SNU-ίδ, A2058, MCF7, SW480. Characterization of the receptor
phosphorylation levels of EGFR, HER2 and HER3 in these 73 ceil lines using PathScan RTK Signaling Antibody Arrays (Cell Signaling Technology) demonstrated elevated HER family activation (Figure 22) .
Effects of antibody treatments in over 70 cancer cell lines (out of more than 100 cell lines tested) on metabolic activity were determined after 96 hours incubation using a similar WST-1 assay as described in Example 2. Results showed that Pan - HER is broadly inhibitory in a large number of cancer cell lines of different tissue origin and genetic background in the presence of Heregulin (Figure 24), EGF (Figure 25), or neither ligand (Figure 23) . "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR -HERS" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082. These results further demonstrated that simultaneous targeting of three receptors provided broader efficacy than targeting of a single receptor or any combination of two receptors in the HER family.
E AMPLE 4: Pan-HER effectively inhibits Hig rsd-iirs lisced proisferatiors
To determine if antibody mixtures are efficacious in the presence of EGFR and HER3 ligands, antibodies mixtures against one, two or three HER family receptors were tested for their ability to inhibit the growth and proliferation of pancreatic cancer cell lines in the presence of Heregulin, EGF, or neither ligand . The effects of treatment with Pan-HER (a mixture of six monoclonal antibodies against EGFR, HER2 and HERS; antibodies 1277, 1565,
4384, 4517, 5038, and 5082), antibody mixtures targeting two HER family members (i .e., EGFR and HER2, EGFR and HERB, and HER2 and HERB), and antibody mixtures targeting one HER family member (i.e., EGFR, HER2 and HERB) were measured on the following ceil lines: CAPAN-1, PK-1, CFPAC-1, BxPC 3, ASPCl, CAPAN-2, PancOS.lB, PANC-1, KP4, MiaPaca-2 and PSN1). The mutation status of these ceil lines is shown in Figure 26. The ability of antibodies mixtures against one, two or three HER family receptors to inhibit the growth and proliferation of a wide variety of cancer cell lines in the presence of Heregulin, EGF, or neither iigand was also tested (Figures 23-25). Cells were exposed to medium containing antibodies and ligands for 96 hours (Iigand and antibody were added simultaneously to the cells). Metabolic activity was determined after 96 hours incubation using a similar WST-1 assay as described in Example 2. "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR-f HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
"EGFR+HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082. Pan-Her exhibited effective- inhibition of a wide variety of cancer cell lines in the presence of EGF or Heregulin.
EXAMPLE 5; Pan-H ER maintains inhibitory effect m cells with acquired resistance to approved therapeutic antibodies
Pan-HER (a mixture of six monoclonal antibodies against EGFR, HER2 and HER3; antibodies 1277, 1565, 4384, 4517, 5038, and 5082) was tested for its ability to inhibit the growth and proliferation of HNS, OE19 and DA-MB-175-VII cell lines or cell pools with acquired resistance to cetuximab, trastuzumab, or pertuzumab, respectively. Cetuximab resistant HNS cell lines were generated as described in Example 11. Trastuzumab resistant OE19 cells and pertuzumab resistant DA-MB-175-VII ceils were established by exposing parental ceils to increasing concentrations of trastuzumab [10- 100 pg/ml] and pertuzumab [1-50 pg/mi] respectively, during a period of eight months and 12 months respectively. Ceils were split once or twice a week in order to keep ceils in expotential growth. The level of resistance was tested every month in a WST-1 viability assay as described in Example 2, until a pool of resistant cells was established. Single cell clones of trastuzumab resistant OE1.9 cells were generated through limited dilution cloning of the acquired trastuzumab resistant pool of OE19 cells as described in Example 11.
Metabolic activity was determined after 96 hours incubation using a similar WST-1 assay as described in Example 2. Pan-HER-treated resistant cells as well as parental cells exhibited reduced levels of metabolic activity (Figure 28) . in contrast, metabolic activity was reduced in parental HN5, OE19 and MDA- B-175-VII cells, but unaltered in resistant clones treated with cetuximab, trastuzumab, or pertuzumab, respectively. This example demonstrates that Pan- HER maintains inhibitory effect in cells with acquired resistance to approved therapeutic antibodies. EXAMPLE 6; Pare-H E effectively prevents compensatory receptor up-regullatsors in vstro
To determine if compensatory receptor up-regulation occurs as a result of treatment with antibody mixtures of the present invention, EGFR, HER2 and HERB levels were measured in whole ceil lysates from H292 and OVCAR8 cell lines after antibody treatment (2.0 pg/ml) for 48 hour by western blot analysis. The effects of treatment with Pan-HER (a mixture of six monoclonal antibodies against EGFR, HER2 and HER3; antibodies 1277, 1565, 4384, 4517, 5038, and 5082) and antibody mixtures targeting one HER family member (i.e., EGFR
(antibodies 1277, 1565, or 1277+1565), HER2 (antibodies 4384, 4517, or 4384+4517) and HER3 (antibodies 5038, 5082, or 5038+5082)) were determined. β-Actin was used as a loading control. Results showed that anti-EGFR treatment lead to HER2 upreguiation in H292 cells (Figure 29, top; cetuximab lane, 1277, 1565, and 1277 +1565 lanes), and anti-HER3 treatment lead to HER2 up-regulation in OVCAR-8 (Figure 29, bottom; M -121 lane, 5038, 5082, and 5038 + 5082 lanes), while Pan-HER treatment lead to the downreguiation of EGFR, HER2 and HER3 (Figure 29; Pan-Her lanes). These results demonstrated that Pan-HER effectively induced simultaneous down-regulation of all three targets and prevented compensatory receptor up-regulation, a potential mechanism for acquiring resistence.
EXAM PLE 7; Synergistic effect of targeting multiple H ER family receptors in BxPC~3 (pancreatic cancer) xenograft model
To evaluate the efficacy of antibody mixtures against EGFR, HER2, HER3 and combinations of two and three receptor targets in xenograft model of human cancer, BxPC-3 (pancreatic cancer) xenograft models were treated with antibody mixtures and the effect on tumor size assayed.
in this assay, BxPC-3 pancreatic cancer cells were inoculated into mice, in brief, 5x106 BxPC3 cells were inoculated subcutaneously into the left flank of eight to ten week old female athymic nude mice. Tumors were measured thrice weekly with calipers and tumor volume in mm3 was calculated according to the formula : (width)2 x length x 0.5. At an average tumor size of 140 mm3 the mice were randomized and treatment initiated. The mice were treated with thrice weekly intraperitoneal injections of 50 mg/kg (10 injections in total) followed by an observation period. Graphical representations of tumor volume data were presented as means ± SEM.
Results showed that Pan-HER (antibodies 1277, 1565, 4384, 451.7, 5038, and 5082) effectively suppressed tumor growth in the BxPC-3 xenograft model (Figure 30; =7/group; treatment period indicated by the light grey area on the graph). A clear synergy was observed when targeting EGFR and HER3 as well as EGFR and HER2, with the former combination being most efficient at controlling growth of the BxPC3 tumor xenografts. "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and
4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR+HER3" refers to a mixture of antibodies 1277., 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082. "Pan-HER" refers to a mixture of antibodies 1.277, 1565, 4384, 4517, 5038, and 5082. Furthermore, EGFR and HER2. down-regulation by Pan-HER in vivo was confirmed by immunohistochemistry on tissue sections from tumors resected 3 days after withdrawal of treatment (Figure 31).
EXAMPLE 8; Synergistic effect of targeting multiple HER family receptors in€aiu~3 (NSCLC) xenograft modei
To evaluate the efficacy of antibody mixtures against EGFR, HER2, HER3 and combinations of two and three receptor targets in xenograft models of human cancer, the Calu-3 (NSCLC) xenograft model were treated with antibody mixtures and the effect on tumor size assayed. In this assay, Calu-3 NSCLC cells were inoculated into mice. In brief, 1x107 Calu-3 cells were inoculated subcutaneousiy into the left flank of eight to ten week old female athymic nude mice. Tumors were measured thrice weekly with calipers and tumor volume in mm3 was calculated according to the formula : (width)2 x length x 0.5. At an average tumor size of 170 mm3 the mice were randomized and treatment initiated. The mice were treated with thrice weekly intraperitoneal injections of 50 mg/kg (8 injections in total). Graphical representations of tumor volume data were presented as means ± SEM.
Results showed that Pan-HER (antibodies 1277, 1565, 4384, 4517, 5038, and 5082) effectively suppressed tumor growth in the Calu-3 xenograft model (Figure 32; N = 5/group; treatment period indicated by the light grey area on the graph). The results show a synergistic effect of targeting EGFR, HER2 and HER3 simultaneuous!y whereas no clear synergy could be observed when targeting EGFR and HER2 or EGFR and HER3 compared to the anti-tumor response of EGFR mono-targeting. "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR+HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
E AMPLE 9: Pan-HER effectsveiy prevents compensatory receptor up-regullatsors in vivo
To determine if prevention of compensatory receptor up-regulation occurs in vivo as a result of treatment with antibody mixtures of the present invention, EGFR, HER2 and HERS levels were measured in antibody-treated BxPC-3 tumor iysates by Western Blot analysis. The effects of treatment with Pan-HER (a mixture of six monoclonal antibodies against EGFR, HER2 and HERS; antibodies 1277, 1565, 4384, 4517, 5038, and 5082), antibody mixtures targeting two HER family members (i.e., EGFR and HER2, EGFR and HER3, and HER2 and HER3), and antibody mixtures targeting one HER family member (i.e., EGFR, HER2 and HER3) were determined. β-.Aetin was used as a loading control. Results showed that anti-EGFR treatment lead to EGFR downreguiation (Figure 33 top), anti-HER2 treatment lead to HER2.
downregu!ation (Figure 33 top)., and anti-HER3 treatment lead to HERS downreguiation (Figure 33 top) . Relative quantification of the Western biot band intensities showed that HER2 was significantly up-regulated in response to anti-HER3 treatment (Figure 33; bottom) . In contrast, Pan-HER treatment resulting in the simultaneous and effective downreguiation of EGFR, HER2 and HER3 (Figure 33 top; green boxes and Figure 33 bottom) . "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. "EGFR+HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517. "EGFR-f HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082. "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
This example demonstrated that Pan-HER is capable of effectively inducing simultaneous down-regulation of ail three targets and preventing compensatory receptor up-regulation in vivo.
EXAMPLE ID: Synergistic effect of targeting multiple HER family receptors in patient- derived AS mutated pancreatic tumor xenograft modeis
To evaluate the in vivo efficacy of antibody mixtures against EGFR, HER2, HER3 and combinations of two and three receptor targets, patient-derived tumor xenograft models of KRAS mutated pancreatic cancer (START Discovery, San Antonio, TX) were treated with antibody mixtures and the effect on tumor size assayed.
in this assay, patient-derived pancreatic cancer cells were inoculated into mice, in brief, viable resected patient tumor material was implanted in immunocompromised mice and serially passaged in vivo. At a tumor volume of 100-200 mm3, animals were randomized into treatment and control groups and dosing was initiated. Dosing schedule: 50 mg/kg i.p. three times weekly, 10 doses in total (day 0-20). N = 5/group. Data are presented as means ± SEM. Asterix indicates first day with p<0.05. The statistically significant difference in treatment response between the groups was maintained throughout the study period.
Results showed that Pan-HER effectively suppressed tumor growth in hard-to-treat patient- derived models of pancreatic cancer. (Figure 34; N = 5/group). Furthermore, deconvolution studies revealed strong synergy of EGFR and HER2 targeting in the ST179 xenograft model and of EGFR and HER3, and to a lesser extent of EGFR and HER2, targeting in the ST383 xenograft model (Figure 35; N = 7-8/group; treatment period indicated by the light grey area on the graph). "EGFR" refers to a mixture of antibodies 1277 and 1565. "HER2" refers to a mixture of antibodies 4384 and 4517. "HER3" refers to a mixture of antibodies 5038 and 5082. " EGFR -HER2" refers to a mixture of antibodies 1277, 1565, 4384, and 4517.
"EGFR+HER3" refers to a mixture of antibodies 1277, 1565, 5038, and 5082. "HER2+HER3" refers to a mixture of antibodies 4384, 4517, 5038, and 5082, "Pan-HER" refers to a mixture of antibodies 1277, 1565, 4384, 4517, 5038, and 5082.
Table 7: Patient-derived xenograft models of pancreatic eaocer
Figure imgf000073_0001
wt: wiid-type,. PD: Progressive disease, ND: Not determined.
E AMPLE 11: Acquired Cetuximab resistant HNS clones show decreased total levels of EGFR together with high EGFR activity
Cetuximab resistant HN5 clones were established by exposing parental H 5 cells to increasing concentraions of cetuximab [1-100 jjg/mi] during a period of six months. Cells were split twice a week in order to keep cells in expotential growth. The level of resistance to cetuximab was tested every month in a WST-1 viability assay as described in Example 2 until a pool of cetuximab resistant cells was established. Single ceil clones were generated through limited dilution cloning of the acquired cetuximab resistant pool of HNS cells. 0.5 cells/well were plated in 384 well plates. Growth and proliferation of single cell colonies was followed using Novartis Ceilavista Imager. The most resistant individual clones, HN5 CR2, HNS CR6, HNS CR13, and HNS CR14, were selected for further characterization (Figure 36).
Viabilty:
The level of cetuximab resistance of individual clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 was tested in a WST-1 viability assay as previously described in Example 1. Briefly, cells were treated with cetuximab at a range of concentrations and assayed 96 hours later. Unlike parental NHS cells, resistant clones were viable with increasing concentrations of cetuximab treatment (Figure 37).
Cetuximab binding to fixed ceils:
The binding strength of cetuximab to parental HNS and resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 was determined. Binding curves were generated by plotting fluorescence signals that were normalized to the number of ceils (DRAQ-5 staining) and cetuximab concentrations. The results demonstrate that while half-maximal binding (i.e., EC50 value) of cetuximab was unaltered, maximal binding was decreased in the resistant clones compared to parental cells (Figure 38) .
EGFR Expression and Signaling :
The relative surface levels of EGFR were determined in parental HNS and resistant ciones HNS CR2, HNS CR6, HNS CR1.3, and HNS CR14. Briefly, ceils were stained with anti-EGFR- FITC (abeam, #11400) or an isotype control (abeam, # 18446) and the relative fluorescence of live ceils quantified by flow cytometry, The relative surface levels of EGFR were lower in cetuximab resistant HNS clones compared to the parental cells (Figure 39) .
The response of cetuximab resistant ciones to EGF stimulation was tested . The total levels of EGFR, levels of phosphorylated EGFR and downstream signaling molecules were determined in parental HN5 and resistant clones HNS CR2, HNS CR6, HNS CR13, and H 5 CR14. Parental HNS ceils and cetuximab resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14 were untreated or stimulated with InM EGF for 15 min before harvesting. Lysates were fractionated on SDS-PAGE followed by Western Blotting for EGFR, the phosphorylated EGFR species pEGFR (Tyrl068), pEGFR(Tyrl045), pEG FR(Ty r 11 3), pEGFR(Tyr992), pEGFR(Thr669), and pEGFR(Serl046/1047), and the signalling molecules AKT, pAKT (Ser473), ERK1/2, pERKl/2(Thr202/Tyr204) (Figures 40 and 41) . β-Actin was used as a loading control . Results showed that the total levels of EGFR and phosphorylated EGFR were lower in cetuximab resistant HNS clones compared to the parental cells (Figure 40) . The results also showed a decreased level of pEGFR(Serl046/1047) in the cetuximab resistant clones, indicating that the feedback mechanism regulating EGFR is less active in the cetuximab resistant clones (Figure 40) . Stimulation with EGF induced a stronger activation of pAKT and pERKl/2 in the cetuximab resistant clones compared to parental HNS cells (Figure 41) . Together, these results demonstrate that EGFR is still active in the cetuximab resistant clones, although EGFR expression is decreased compared to parental HNS cells.
E AMPLE 12; Antibody mixtures targeting EGFR overcome cetisximab resistance through efficient EGFR srsterrsaii^atsori followed by degradation of the receptor
A mixture of antibodies targeting non-overlapping epitopes on EGFR was tested for its abliiity to partially overcome the cetuximab induced resistance in cetuximab resistant HNS clones HNS CR2 and HNS CR1.4. Parental HNS, HNS CR2, and HNS CRM ceils were treated with EGFR-LNA™ (EGFR targeting Locked Nucleic Acid, Exiqon), Cetuximab or EGFR-2mix (antibodies 1277 and 1565) for 48 hours. Growth and proliferation was measured using a WST- 1 assay as described in Example 2 and quantification of the effects were plotted with data points representing a mean +/- SEM (n=4) (Figure 42) . EGFR-LNA, and EGFR-2mix both induced a similar reduction in ceil viability. These results demonstrated that a mixture of antibodies targeting non-overlapping epitopes on EGFR partially overcame the cetuximab induced resistance and that the cetuximab resistant clones remain dependent on EGFR for growth and proliferation (Figure 42).
The levels of EGFR in cells treated with EGFR-LNA, cetuximab or EGFR-2mix for 43 hour was determined by fractionating cellular lysates on a SDS-PAGE followed by Western Blotting for EGFR. The results showed that efficient EGFR internalization followed by lysosomal degradation of the receptor was induced in antibody treated resistant cells (Figure 43), and thus providing a mechanism for the ability of the antibody mixture targeting EGFR overcome cetuximab resistance.
EXAM PLE 13; Cetuximab resistant H US cios es escape treatment through H ERS arid IGFI R
The observed level of inhibition of the resistant clones by the anti-EGFR mixture did not, however, induce as efficient growth inhibition as in the parental cells, suggesting that alternative receptor tyrosine kinases (RTKs) may be involved in the mechanism of acquired cetuximab resistance. To test the role of HERS activity in parental NHS and resistant clones HNS CR2, HNS CR6, HNS CR13, and HNS CR14, ceils were treated with a mixture of two EGFR antibodies (antibodies 1277 and 1565), a mixture of two HER3 antibodies (antibodies 5038 and 5082), a mixture of two EGFR and two HER3 antibodies (antibodies 1277, 1565, 5038 and 5082), or cetuximab for 48 hours. Growth and proliferation was measured using a WST-1 assay as described in Example 2 and quantification of the effects were plotted with data points representing as a mean +/■■ SEM (n=6) . The results showed superior effects of the mixture containg antibodies targeting both HERB and EGFR compared to effects induced by the EGFR antibody mixture alone. These results support the hypothesis of involvement of alternative RTKs in the acquired cetuximab resistance (Figure 44) . The dose response curves of parental HNS and resistant HNS CR2 cells to the antibody mixtures are shown in Figure 45A and B.
The involvement of HER3 in the acquired resistance to cetuximab shown here indicates the plasticity of the RTK family as a mechanism of acquired resistance to cetuximab in vitro.
Table 8; Sequences of selected chimeric antibodies
Antibody 1277: VH nucleotide sequence
cgcgccgaag tgcagctggt ggagtctggg ggaggcttag tgaagcctgg agagtccttg
aaactctcct gtgcagcctc tggattcgct ttcagttact ctgacatgtc ttgggttcgc
cagactccgg agaagaggct ggagtgggtc gcatacatga gtagtgctgg tgatgtcacc
ttctattcag acactgtgaa gggccgattc accatctcca gagacaatgc caagaacacc
ctgtatctgc aagtgagcag tctgaagtct gaggacacag ccatatatta ctgtgtaaga
caccgggacg tggctatgga ctactggggt caaggaacct cagtcaccgt ctcg (SEQ ID NO: 14)
Antibody 1277: VH amino acid sequence
Arg Ala Glu Vai Gin Leu Vai Giu Ser Gly Giy Gly Leu Val Lys Pro G!y Glu Ser Leu Lys Leu Ser Cys Ala Ala Ser G!y Phe Ala Phe Ser
Tyr Ser Asp Met Ser Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu
Trp Val Ala Tyr Met Ser Ser Ala Gly Asp Val Thr Phe Tyr Ser Asp
Thr Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr Leu Gin Val Ser Ser Leu Lys Ser Glu Asp Thr Ala lie Tyr
Tyr Cys Val Arg His Arg Asp Val Ala Met Asp Tyr Trp Gly Gin Gly
Thr Ser Val Thr Val Ser (SEQ ID NO: 15)
Antibody 1277: light chain nucleotide sequence
ctagccgatg ttgtgatgac ccagactcca ctctccctgc ctgtcagtct tggagatcaa
gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta
cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac
cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc
aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aagtacacat
gttccgacgt tcggtggagg caccaagctg gaaatcaaac gaactgtggc tgcaccatct
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc
gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt (SEQ ID NO: 16
Antibody 1277: light chain amino acid sequence
Leu Ala Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser
Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Val
His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly
Gin Ser Pro Lys Leu Leu He Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys He Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser
Gin Ser Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He
Lys Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp
Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu
Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys (SEQ ID NO: 17)
Antibody 1565: VH nucleotide sequence
ggcgcgccga ggtccaactg caacagtctg ggactgaatt ggtgaagcct ggggcttcag
tgatactgtc ctgtaaggcc tctggctaca ccttcaccag ctactggatg cagtgggtga agcagaggcc tggacaaggc cttgagtgga ttggaaatat taatcctagc aatggtggaa ctagtttcaa tgaggagttc aagagtaggg ccacactgac tgtagacaaa tcctccagta cagcctacat gcaactcagc agcctgacat ctgaggactc tgcggtctat tattgtgcaa gagacggggg cctttacgac ggatactact ttgacttctg gggccaaggc accactctca cagtctcgag (SEQ ID NO: 18)
Antibody 1565 : VH amino acid sequence
Arg Aia G!u Val Gin Leu Gin Gin Ser Giy Thr Giu Leu Val Lys Pro Giy Ala Ser Vai He Leu Ser Cys Lys Aia Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gin Tip Val Lys Gin Arg Pro Gly Gin Gly Leu Giu Trp He Giy Asn lie Asn Pro Ser Asn Gly Gly Thr Ser Phe Asn Giu Giu Phe Lys Ser Arg Aia Thr Leu Thr Vai Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Giu Asp Ser Ala Val Tyr Tyr Cys Aia Arg Asp Giy Giy Leu Tyr Asp Giy Tyr Tyr Phe Asp Phe Trp Giy Gin Gly Thr Thr Leu Thr Vai Ser (SEQ ID NO : 19)
Antibody 1565 : light chain nucleotide sequence
gctagccaac attgtgatga cacagtctca caaattcatg tccacattaa taggagccag ggtctccatc acctgcaagg ccagtcagga tgtggatacg gctgtagcct ggtatcaaca gaaaccaggt caatctccta aattattaat ttattgggca tccacccggc acactggagt ccctgatcgc ttcacaggca gtggatctgg gacagatttc tctctcaccg ttagcaatgt gcagtctgag gacttaacag attatttctg tcagcaatat agcagctatc ctctcacgtt cggtgctggg accaagctgg agctgaaacg aactgtggct gcaccatctg tcttca ctt cccgccatct gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg ggagagtgtt aataagcggc cgc (SEQ ID NO : 20)
Antibody 1565 : light chain amino acid sequence
Leu Aia Asn lie Vai Met Thr Gin Ser His Lys Phe Met Ser Thr Leu
He Giy Aia Arg Val Ser lie Thr Cys Lys Ala Ser Gin Asp Val Asp
Thr Aia Vai Aia Trp Tyr Gin Gin Lys Pro Giy Gin Ser Pro Lys Leu
Leu He Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Thr Giy Ser Giy Ser Giy Thr Asp Phe Ser Leu Thr Vai Ser Asn Vai
Gin Ser Giu Asp Leu Thr Asp Tyr Phe Cys Gin Gin Tyr Ser Ser Tyr
Pro Leu Thr Phe Gly Ala Giy Thr Lys Leu Giu Leu Lys Arg Thr Vai
Aia Ala Pro Ser Vai Phe lie Phe Pro Pro Ser Asp Giu Gin Leu Lys
Ser Gly Thr Aia Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg G!u Ala Lys Va! Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Giy Asn Ser Gin Giu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Giu Lys His Lys
Val Tyr Ala Cys Giu Val Thr His Gin Giy Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn Arg Giy Giu Cys (SEQ ID NO: 21)
Antibody 4384: VH nucleotide sequence
caggtgcagc tgcagcagcc tggcacaqaq ctggtgaaac ctggcgcctc cgtgaagctg tcctgcaagg cctccggcta caccttcacc tcccactgga tgcactgggt gaaacagcgg cctqqacagg gcctggaatg gatcggcaac atcaacccct ccaacggcqg caccaactac aacgagaagt tcaagtcccg ggccaccctg accgtggaca aggcctcctc caccgcctac atgcagctgt cctccctgac ctccgaggac tccgccgtgt actactgcgc cagaqcctac
tacgacttca gttggttcgt gtactggggc cagggcaccc tggtgacagt ctcg (SEQ ID NO: 22)
Antibody 4384: VH amino acid sequence
Gin Val Gin Leu Gin Gin Pro Giy Thr Giu Leu Val Lys Pro Giy Ala
Ser Val Lys Leu Ser Cys Lys Ala Ser Giy Tyr Thr Phe Thr Ser His
Trp Met His Trp Val Lys Gin Arg Pro Giy Gin Giy Leu Glu Trp lie
Giy Asn lie Asn Pro Ser Asn Giy Giy Thr Asn Tyr Asn Glu Lys Phe
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ala Ser Ser Thr Ala Tyr
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
Ala Arg Ala Tyr Tyr Asp Phe Ser Trp Phe Vai Tyr Trp Giy Gin Giy
Thr Leu Vai Thr Val Ser (SEQ ID NO: 23)
Antibody 4384: light chain nucleotide sequence
gatatccaga tgacccagac ctcctccagc ctgtccgcct ccctgggcga cagagtgacc atctcctgcc ggtcctccca ggacatctcc aactacctga actggtatca gcagaaaccc
gacggcaccg tgaagctgct gatgtacatc tcccggctgc actccggcgt gccctccaga ttctccggct ctggctccgg caccgagtac tccctgacca tcagcaacct ggaacaggaa gatatcgcta cctacttctg tcagcagggc aacaccctgc ccctgacctt cggcgctggc
accaagctgg aactgaagcg gaccgtggcc gctccctccg tgttcatctt cccaccctcc
gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaacaa cttctacccc cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa ctcccaggaa tccgtgaccg agcaggactc caaggacagc acctactccc tgtcctccac cctgaccctg tccaaggccg actacgagaa gcacaaggtg tacgcctgcq aagtgaccca ccagggcctg tccagccccg tgaccaagtc cttcaaccgg ggcgagtgc (SEQ ID NO: 24)
Antibody 4384: light chain amino acid sequence
Asp lie Gin Met Thr Gin Thr Ser Ser Ser Leu Ser Ala Ser Leu Giy
Asp Arg Vai Thr He Ser Cys Arg Ser Ser Gin Asp He Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp G!y Thr Vai Lys Leu Leu Met Tyr He Ser Arg Leu His Ser Giy Vai Pro Ser Arg Phe Ser Gly Ser
Giy Ser Giy Thr Giu Tyr Ser Leu Thr lie Ser Asn Leu Giu Gin Giu
Asp lie Aia Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Leu Thr
Phe Gly Ala Gly Thr Lys Leu Giu Leu Lys Arg Thr Vai Ala Aia Pro
Ser Vai Phe He Phe Pro Pro Ser Asp Giu Gin Leu Lys Ser Giy Thr
Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Giu Ala Lys
Va Gin Trp Lys Vai Asp Asn Aia Leu Gin Ser Gly Asn Ser Gin Giu
Ser Vai Thr Giu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Aia Asp Tyr Giu Lys His Lys Vai Tyr Ala
Cys Giu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser Phe
Asn Arg Gly Giu Cys (SEQ ID NO: 25)
Antibody 4517: VH nucleotide sequence
gaagtgcagc tggtggaatc tggcggcgac ctggtgaaac ctggcggctc cctgaagctg tcctgcgccg cctccggctt caccttctcc agctacggca tgtcctgggt gcgactgacc
cccgacaagc ggctggaatg ggtggcaacc atctccggcg gaggctccta cacctactac cccgactccg tgaagggccg gttcaccatc tcccgggata tcgccaagtc caccctgtac
ctgcagatgt cctccctgaa gtccgaggac accgccgtgt actactgcgc ccggaagggc
aactacggca attacggcaa gctggcctac tggggccagg gcacctccgt gacagtctcg (SEQ ID NO
Antibody 4517: VH amino acid sequence
Giu Vai Gin Leu Vai Giu Ser Giy Gly Asp Leu Vai Lys Pro Gly Giy
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
Giy Met Ser Trp Vai Arg Leu Thr Pro Asp Lys Arg Leu Giu Trp Vai
Ala Thr He Ser Giy Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Vai
Lys Giy Arg Phe Thr He Ser Arg Asp lie Aia Lys Ser Thr Leu Tyr
Leu Gin Met Ser Ser Leu Lys Ser Giu Asp Thr Aia Vai Tyr Tyr Cys
Aia Arg Lys Gly Asn Tyr Gly Asn Tyr Gly Lys Leu Ala Tyr Trp Giy
Gin Giy Thr Ser Vai Thr Vai Ser (SEQ ID NO: 27)
Antibody 4517: light chain nucleotide sequence
gatatccaga tgacccagtc ccccgcctcc ctgtccgtgt ctgtgggcga gacagtgacc
atcacctgtc gggcctccga gaacatctac tccaacctgg cctggtatca gcaggaacag
ggcaagtccc cccagctgct ggtgtacgcc gccaccaatc tggccgacgg cgtgccctcc
agattctccg gctctggctc cggcacccag tactccctga agatcaactc cctgcagtcc
gaggacttcg gctcctacta ctgccagcac ttctggggca ccccctggac cttcggcgga
ggcaccaagc tggaaatcaa gcggaccgtg gccgctccct ccgtgttcat cttcccaccc
tccgacgagc agctgaagtc cggcaccgcc tccgtggtgt gcctgctgaa caacttctac
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caactcccag gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gc (SEQ ID NO: 28)
Antibody 4517: light chain amino acid sequence
Asp lie Gin Met Thr Gin Ser Pro Aia Ser Leu Ser Vai Ser Vai Giy Giu Thr Val Thr He Thr Cys Arg Ala Ser Giu Asn lie Tyr Ser Asn Leu Aia Trp Tyr Gin Gin Giu Gin Giy Lys Ser Pro Gin Leu Leu Val Tyr Ala Ala Thr Asn Leu Ala Asp Giy Val Pro Ser Arg Phe Ser Giy Ser Giy Ser Giy Thr Gin Tyr Ser Leu Lys lie Asn Ser Leu Gin Ser Giu Asp Phe Giy Ser Tyr Tyr Cys Gin His Phe Trp Giy Thr Pro Trp Thr Phe Giy Giy Giy Thr Lys Leu Giu He Lys Arg Thr Vai Aia Aia Pro Ser Vai Phe lie Phe Pro Pro Ser Asp Giu Gin Leu Lys Ser Giy Thr Aia Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Giu Aia Lys Vai Gin Trp Lys Vai Asp Asn Aia Leu Gin Ser Giy Asn Ser Gin Giu Ser Val Thr Giu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Aia Asp Tyr Giu Lys His Lys Vai Tyr Aia Cys Giu Val Thr His Gin Giy Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Giy Giu Cys (SEQ ID MO: 29)
Antibody 5038: VH nucleotide sequence
cgcgccgagg tgaagctggt tgagtcagga cctggcctcg tgaaaccttc tcagtctctg tctctcacct gctctgtcac tggctactcc atcaccagtg gtttttactg gacctggatc cggcagtttc caggcaacaa attggaatgg a gggcttca taagctacga tgg agcaat aactacaacc catctctcaa aaatcgaatc tccatcactc gtgacacatc taagaaccag tttttcctga agttgaattc tgtgactact gaggacacag ccacatatta ctgtgcaaga ggcggaggct actatggtaa cctctttgac tactggggcc aaggcaccac tctcacagtc tcga (SEQ ID NO: 30)
Antibody 5038: VH amino acid sequence
Arg Ala Giu Val Lys Leu Vai Giu Ser Giy Pro Giy Leu Val Lys Pro Ser Gin Ser Leu Ser Leu Thr Cys Ser Val Thr Giy Tyr Ser He Thr Ser Giy Phe Tyr Trp Thr Trp lie Arg Gin Phe Pro Giy Asn Lys Leu Giu Trp Met Giy Phe He Ser Tyr Asp Giy Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn Arg lie Ser lie Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Giu Asp Thr Ala Thr Tyr Tyr Cys Aia Arg Giy Giy Giy Tyr Tyr Giy Asn Leu Phe Asp Tyr Trp Giy Gin Giy Thr Thr Leu Thr Val Ser (SEQ ID NO: 31)
Antibody 5038: light chain nucleotide sequence ctagccgata ttgtgatgac tcaaactaca tcctccctgt ccgcctctct gggagacaga
gtcaccatca gttgcaggcc aagtcaggac attagcaatt atgtaaactg gtttcagcag
aaaccaggtg gaactgttaa gctcctgatc ttccacacat caagattaca ctcaggagtc
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaccctg
gaacaggaag atattgccat ttacttttgc caacagggta ttacgcttcc gtggacgttc
ggtggcggca ccaagctgga aataaaacga actgtggctg caccatctgt cttcatcttc
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta ataagcggcc (SEQ ID NO: 32)
Antibody 5038: light chain amino acid sequence
Leu Ala Asp∑!e Vai Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser
Leu Gly Asp Arg Va! Thr He Ser Cys Arg Pro Ser Gin Asp lie Ser
Asn Tyr Val Asn Trp Phe Gin Gin Lys Pro Giy Gly Thr Vai Lys Leu
Leu lie Phe His Thr Ser Arg Leu His Ser Giy Vai Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ser Thr Leu
Giu Gin Glu Asp He Aia lie Tyr Phe Cys Gin Gin Gly He Thr Leu
Pro Trp Thr Phe Gly Giy Gly Thr Lys Leu Glu He Lys Arg Thr Val
Ala Aia Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Giu Ala Lys Vai Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn
Ser Gin Glu Ser Vai Thr Giu Gin Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys (SEQ ID NO: 33)
Antibody 5082: VH nucleotide sequence
cgcgccgagg tgcagctgaa ggagtcagga cctggcctcg tgaaaccttc tcagtctctg
tctctcacct gctctgtcac cggctactcc atcaccagtg cttattactg gaactggatc
cggcagtttc caggaaacaa agtggaatgg atgggctaca taggctacga tggtcgtaat
acctacaacc catctctcaa aaatcgaatc tccatcactc gtgacacatc taagaaccag
tttttcctga aattgaattc tctgactact gaggacacag ccacatatta ttgttcaaga
gagggggact acggttactc tgactactgg ggccaaggca ccactctcac agtctcga (SEQ ID NO: 34)
Antibody 5082: VH amino acid sequence
Arg Ala Glu Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gin Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser He Thr
Ser Ala Tyr Tyr Trp Asn Trp He Arg Gin Phe Pro Gly Asn Lys Vai G!u Trp Met G!y Tyr He Giy Tyr Asp Giy Arg Asn Thr Tyr Asn Pro
Ser Leu Lys Asn Arg He Ser lie Thr Arg Asp Thr Ser Lys Asn Gin
Phe Phe Leu Lys Leu Asn Ser Leu Thr Thr Giu Asp Thr Ala Thr Tyr
Tyr Cys Ser Arg Giu Giy Asp Tyr Gly Tyr Ser Asp Tyr Trp G!y Gin
Giy Thr Thr Leu Thr Va! Ser (SEQ ID NO: 35)
Antibody 5082: light chain nucleotide sequence
ctagccgata ttgtgatgac gcaagctaca tcctccctgt ctgcctctct gggagacaga
gtcaccgtca gttgcagggc aagtcaggac attaacaatt atttaaattg gtatcagcag
aagccagatg gaactgttaa actcctgatc tactacacat caagattaca gtcaggagtc
ccatcaaggt tcagtggcag tgggtctgga atagattatt ctctcaccat tagcaacctg
gagcaggaag attttgtcac ttacttttgc caacagagtg aaacgcttcc gtggacgttc
ggtggaggca ccaagctgga gctgaaacga actgtggctg caeca tctgt cttcatcttc
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac
ttctatccca gagaggecaa agtacagtgg aaggtggata acgccctcca ategggtaac
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat
cagggectga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta ataagcggcc (SEQ ID NO: 36)
Antibody 5082: light chain amino acid sequence
Leu Ala Asp lie Val Met Thr Gin Ala Thr Ser Ser Leu Ser Ala Ser
Leu G!y Asp Arg Val Thr Va! Ser Cys Arg Ala Ser Gin Asp lie Asn
Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu
Leu lie Tyr Tyr Thr Ser Arg Leu Gin Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly He Asp Tyr Ser Leu Thr lie Ser Asn Leu
Giu Gin Giu Asp Phe Val Thr Tyr Phe Cys G!n Gin Ser Giu Thr Leu
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Giu Leu Lys Arg Thr Val
Ala Aia Pro Ser Val Phe He Phe Pro Pro Ser Asp Giu Gin Leu Lys
Ser Gly Thr Aia Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Giu Aia Lys Vai Gin Trp Lys Vai Asp Asn Aia Leu Gin Ser Gly Asn
Ser Gin Giu Ser Vai Thr Giu Gin Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Giu Lys His Lys
Vai Tyr Ala Cys Giu Vai Thr His Gin Giy Leu Ser Ser Pro Vai Thr
Lys Ser Phe Asn Arg Gly Giu Cys (SEQ ID NO: 37)

Claims

What is claimed is:
1 , A recombinant antibody composition comprising at least one humanized anti- EGFR antibody or an antigen-binding fragment thereof, at least one humanized anti-HER2 antibody or an ant gen-b nding fragment thereof, and at least one humanized anti-HER3 antibody or an antigen-binding fragment thereof.
2, The antibody composition of claim 1, wherein:
the at least one humanized anti-EGFR antibody is selected from
(a) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO: l and the light chain variable region sequence of SEQ ID NQ:3 or SEQ ID NO :2, and
(b) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO:4 and the light chain variable region sequence of SEQ ID NO:5; the at least one humanized anti-HER2 antibody is selected from
(c) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO:6 and the light chain variable region sequence of SEQ ID NO:7, and
(d) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO:8 and the light chain variable region sequence of SEQ ID NO:9; and the at least one humanized anti~HER3 antibody is selected from
(e) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO: 10 and the light chain variable region sequence of SEQ ID NO: l 1, and
(f) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO: 12 and the light chain variable region sequence of SEQ ID NO: 13.
3, The antibody composition of claim 2, comprising
(a) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO:43 and the light chain variable region sequence of SEQ ID NO:44;
(b) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO:47 and the light chain variable region sequence of SEQ ID NQ:48;
(c) an antibody comprising the heavy chain variable region sequence of SEQ
ID NO:51 and the light chain variable region sequence of SEQ ID NO:52; an antibody comprising the heavy chain variable region sequence of SEQ ID NO:53 and the light chain variable region sequence of SEQ ID NO:54; an antibody comprising the heavy chain variable region sequence of SEQ ID NO:55 and the light chain variable region sequence of SEQ ID NO:56; and
an antibody comprising the heavy chain variable region sequence of SEQ I D NO:6! and the light chain variable region sequence of SEQ ID NO: 62.
4. The antibody composition of claim 2, comprising anti-EGFR antibodies (a) (b), anti-HER2 antibodies (c) and (d), and anti-HER3 antibodies (e) and (f).
5. The antibody composition of claim 2, comprising:
(i) anti-EGFR antibody (a), anti-HER2 antibody (c), and anti-HER3 antibody (e);
(ii) anti-EGFR antibody (a), anti~HER2 antibody (c), and anti-HER3 antibody (f);
(iii) anti-EGFR antibody (a), antl-HER2 antibody (d), and anti-HER3 antibody (e);
(iv) anti-EGFR antibody (a), anti-H ER2 antibody (d), and anti-H ER.3 antibody (f);
(v) anti-EGFR antibody (b), anti-HER2 antibody (c), and anti-HER3 antibody (e);
(vi) anti-EGFR antibody (b), anti-HER2 antibody (c), and anti-HER3 antibody (f);
(vii) anti-EGFR antibody (b), anti-HER2 antibody (d), and anti-HER3 antibody (e); or
(viii) anti-EGFR antibody (b), anti-HER2 antibody (d), and anti-HER3 antibody (f).
6. The antibody composition of any of the preceding claims, wherein the anti- EGFR antibody (a) comprises:
(i) a heavy chain variable region sequence comprising SEQ ID NO: l that has Arg44, Val83 and He 104, and a light chain variable region sequence comprising SEQ ID NO:3 that has Leu34, Tyr41 , Leu51 and Phe92;
(ii) a heavy chain variable region sequence comprising SEQ ID NO: l that has Arg44, Val83 and He 104, and a light chain variable region sequence comprising SEQ ID NO:3 that has Tyr41 , Leu51 and Phe92; or
(iii) a heav chain variable region sequence comprising SEQ ID NO: l that has Arg44 and Val83, and a light chain variable region sequence comprising SEQ ID NO:2 that has Ala 19 and Phe92.
7, The antibody composition of any of the preceding claims, wherein the anti- EGFR antibody (b) comprises:
(i) a heavy chain variable region sequence comprising SEQ ID NO:4 that has Leu20, Ile48 and Ala68, and a light chain variable region sequence comprising SEQ ID NO:5 that has Val75 and Phe87; or
(ii) a heavy chain variable region sequence comprising SEQ ID O:4 that has Leu20, Ile48, Leu56, and Ala68, and a light chain variable region sequence comprising SEQ ID NO:5 that has Val75 and Phe87.
8, The antibody composition of any of the preceding claims, wherein the anti- HER2 antibody (c) comprises:
(i) a heavy chain variable region sequence comprising SEQ ID NO: 6 that has Ser55, Leu70, Val72, Lys74 and A la79, and a light chain variable region sequence comprising SEQ ID NO: 7 that has Val44, Met48 and Tyr70; or
(ii) a heavy chain variable region sequence comprising SEQ ID NO:6 that has Ser55 and Val72, and a light chain variable region sequence comprising SEQ ID NO:7 that has Met48 and Tyr70.
9, The antibody composition of any of the preceding claims, wherein the anti- HER2 antibody (d) comprises a heavy chain variable region sequence comprising SEQ ID NO: 8 that has Ala49, Ile74 and Ser77, and a light chain variable region sequence comprising SEQ ID NO:9 that has Thr56, Tyr71, Ser85 and Leu! 04.
10, The antibody composition of any of the preceding claims, wherein the anti- HER3 antibody (e) comprises a heaw chain variable region sequence comprising SEQ ID NO: 10 that has Met49, Ser55 and I!e68, or Asn44, Ser55 and Thr93, and a light chain variable region sequence comprising SEQ ID NO:l 1 that has Phe36, VaI44, Phe49 and Sle85, or has Phe36, Phe49 and Leu73.
1 1 , The antibody composition of any of the preceding claims, wherein the anti- HER3 antibody (f) comprises: (i) a heavy chain variable region sequence comprising SEQ ID NO: 12 that has Val46, Me†49, Ser55 and Arg72, and a light chain variable region sequence comprising SEQ ID NO: 13 that has Val21, Thr29, Val44, and Phe87; or
(ii) a heaw chain variable region sequence comprising SEQ ID NO: 12 that has Phe41, Val46, Met49, Ser55 and Arg72, and a light chain variable region sequence comprising SEQ ID NO: 13 that has Val2I, Val44, Tyr71, Phe87 and Leu 104.
12. The antibody composition of claim 2, comprising three, four, five, or six antibodies.
13. The antibody composition of claim 2, comprising:
(a) anti-EGFR antibody 10292, 10460, or 11294;
(b) anti-EGFR antibody 10560 or 11302;
(c) anti-HER2 antibody 10704 or 1 1249;
(d) anti-HER2 antibody 11145;
(e) anti-HER3 antibody 10738 or 10810; and
(f) aiiti-HER3 antibody 11006 or 11052.
14. A pharmaceutical composition comprising a humanized recombinant antibody composition according to any of claims 1-13 and at least one pharmaceu tically acceptable diluent, carrier or excipient.
15. The phannaceutical composition comprising the antibody composition of claim 3 and at least one pharmaceutically acceptable diluent, carrier or excipient.
16. The pharmaceutical composition of claim 1 or 15, wherein at least one antibody in the composition is an immunoconjugate wherein the antibody is conjugated to an anti-cancer agent.
17. A humanized anti-EGFR antibody whose heavy and light chain amino acid sequences comprise:
SEQ ID NOs:43 and 44, respectively,
SEQ ID NOs:38 and 39, respectively, SEQ ID NOs:41 and 42, respectively,
SEQ ID NOs:45 and 46, respectively, or
SEQ ID Os:47 and 48, respectively,
or an antigen-binding fragment thereof.
18. A humanized anti-HER2 antibody whose heavy and light chain amino acid sequences comprise:
SEQ ID NOs:51 and 52, respectively,
SEQ ID NOs:49 and 50, respectively, or
SEQ ID NOs:53 and 54, respectively,
or an antigen-binding fragment thereof.
19. A humanized anti-HER3 antibody whose heavy and light chain amino acid sequences comprise:
SEQ ID NOs:55 and 56, respectively,
SEQ ID Os:57 and 58, respectively,
SEQ ID NOs:59 and 60, respectively, or
SEQ ID NOs:61 and 62, respectively,
or an antigen-binding fragment thereof.
20. A nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain, or the light chain, or both, of an antibody or antigen-binding fragment of any of claims 17-19.
21. An expression vector comprising a nucleic acid molecule according to claim
20.
22. A host ceil comprising a nucleic acid molecule according to claim 21 or an expression vector according to claim 21, wherein said host cell is capable of expressing the antibody or antigen -bin ding fragment encoded by said nucleic acid molecule.
23. A method for producing an antibody, comprising providmg a host cell according to claim 22, cul tivating said host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody.
24. A method for producing a recombinant antibody composition comprising at least one humanized recombinant anti-EGFR antibody, at least one humanized recombinant anti-HER2 antibody and at least one humanized recombinant anti-HER3 antibody, the method comprising:
providmg at least first, second and third host cells, wherein the first host cell is capable of expressing a recombinant anti-EGFR antibody as defined in claim 17, the second host cell is capable of expressing a recombinant anti-HER2 antibody as defined in claim 18, and the third host cell is capable of expressing a recombinant anti-HE 3 antibody as defined in claim 18,
cultivating the first, second and third host cells under conditions suitable for expression of the anti-EGFR antibody, the anti-HER2 antibody and the anti-HER3 antibody, and
isolating the resulting antibodies.
25. A method for treating cancer in a patient, the method comprising administering to said patient a recombinant antibody composition according to any of claims 1-13 or a pharmaceutical composition according to claim 14 or 15.
26. A method for treating a patient with a disorder characterized by expression or overexpression of EGFR, HER2 and/or HER3, the method comprising administering to said patient a recombinant antibody composition according to any of claims 1-13 or a
pharmaceutical composition according to claim 14 or 15.
27. A method for treating cancer in a patient having acquired resistance to treatment with an antibody and/or a tyrosine kinase inhibitor, the method comprising administering to said patient an effective amount of a recombinant antibody composition according to any of claims 1-13 or a pharmaceutical composition according to claim 14 or 15.
28. A method for inhibiting cancer growth in a patient, the method comprising administering to said patient a recombinant antibody composition according to any of claims 1-13 or a pharmaceutical composition according to claim 14 or 15.
29. A method for reducing EG FR, HER2, or ER3 expression, or preventing EGFR, HER2, or HER3 up-regulation in a cancer patient, comprising administering to the patient a recombinant antibody composition according to any of cl aims 1 -13 or a
pharmaceutical composition according to claim 14 or 15.
30. The method of any one of claims 25-29, wherein the patient has pancreatic, bone, colon, endometrial, or urinary tract cancer.
31. The method of claim 30, wherein the patient has pancreatic cancer and a KRAS mutation,
32. The method of any one of claims 25-31 , wherein at least one of the antibodies is conjugated to an anti-cancer agent.
33. The method of claim 32, wherein the anti-cancer agent is a cytotoxic agent, a cytokine, a toxin, or a radionuclide.
34. The method of any one of claims 25-33, wherein the patient has not been treated for cancer previously.
35. The method of any one of claims 25-33, wherein the patient has been treated for cancer previously.
36. The method of claim 35, wherein the patient has been treated with cetuximab, trastuzumab, or pertuzurnab previously.
37. The method of claim 36, wherein cancer in the patient has acquired resistance to cetuximab, trastuzumab, or pertuzurnab.
PCT/IB2013/001027 2012-05-02 2013-05-02 Humanized pan-her antibody compositions WO2013164689A2 (en)

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CN201380033627.0A CN104428318B (en) 2012-05-02 2013-05-02 The general HER antibody compositions of humanization
CA2872226A CA2872226A1 (en) 2012-05-02 2013-05-02 Humanized pan-her antibody compositions
KR1020147033605A KR101933990B1 (en) 2012-05-02 2013-05-02 Humanized pan-her antibody compositions
EP13729074.8A EP2844675B1 (en) 2012-05-02 2013-05-02 Humanized pan-her antibody compositions
JP2015509512A JP6325527B2 (en) 2012-05-02 2013-05-02 Humanized PAN-HER antibody composition
AU2013255537A AU2013255537B2 (en) 2012-05-02 2013-05-02 Humanized pan-her antibody compositions
MX2014013213A MX358728B (en) 2012-05-02 2013-05-02 Humanized pan-her antibody compositions.
BR112014027291A BR112014027291A2 (en) 2012-05-02 2013-05-02 humanized pan-her antibody compositions
IL235386A IL235386A0 (en) 2012-05-02 2014-10-29 Humanized pan-her antibody compositions
HK15103006.9A HK1202431A1 (en) 2012-05-02 2015-03-24 Humanized pan-her antibody compositions pan-her
US15/350,801 US10058610B2 (en) 2012-05-02 2016-11-14 Methods for treating cancer by administering humanized pan-HER antibody compositions
US16/058,086 US20180344846A1 (en) 2012-05-02 2018-08-08 Humanized pan-her antibody compositions

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