WO2017015008A1 - Anticorps anti-her3 - Google Patents

Anticorps anti-her3 Download PDF

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Publication number
WO2017015008A1
WO2017015008A1 PCT/US2016/041973 US2016041973W WO2017015008A1 WO 2017015008 A1 WO2017015008 A1 WO 2017015008A1 US 2016041973 W US2016041973 W US 2016041973W WO 2017015008 A1 WO2017015008 A1 WO 2017015008A1
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Prior art keywords
antibody
cancer
seq
acid sequence
present
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PCT/US2016/041973
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English (en)
Inventor
Max Ephraim ADER
James D. Pancook
Mark A. WORTINGER
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Eli Lilly And Company
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Publication of WO2017015008A1 publication Critical patent/WO2017015008A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of medicine. More particularly, the present invention relates to antibodies that bind human HERS, and may be useful for treating cancer, especially tumor metastasis, alone or in combination with other antineoplastic agents such as human epidermal growth factor receptor (EGFR) inhibitors for solid tumors driven by EGFR signaling and/or that use HERS as a resistance mechanism, including hepatocellular carcinoma, gastric, lung, head and neck, colorectal, pancreatic, renal or breast cancers.
  • EGFR human epidermal growth factor receptor
  • Dysregulation of the epidermal growth factor family of signaling receptors plays a critical role in the initiation and progression of a variety of human cancers.
  • the EGFR family receptor HERS and its ligand neuregulm 1 (NRG1) play a role in a variety of human malignancies, and act as a major mechanism of de novo or acquired resistance to EGFR inhibition and other targeted therapies.
  • HER3 is the major Akt pathway recruitment center for the EGFR signaling family, and is activated through both ligand dependent (i.e., NRG1) and ligand independent mechanisms.
  • WO 2015/067986 disclosed a murine anti-human-HER3 antibody, named 9F7-F11, having affinity for HER3 that is not inhibited when neuregulin is present (i.e., the 9F7-F1 1 antibody is neuregulin non-competitive), but, like the antibodies in one of the classes of antibodies reported by Vincent, S., et al, 9F7-F1 1/HER3 affinity is also ailosterically increased when neuregulin is present in the environment of HER3 -positive cells (i .e., 9F7-F11 is an allosteric anti-HER3 antibody).
  • the Kd value of 9F7-F 11 binding to HER3 was reported to be 0.47 + 0.07 nM following co-incubation with NRG1 whereas Kd was measured at 2.33 + 0.30 nm without NRG1: thus, demonstrating that NRGl addition allosterically induces a 6-fold increase of 9F7-F11 affinity to the HERS receptor.
  • HER3 EGFR pathway by binding human HER3 with high affinity which is allosterically increased when neuregulin is present in the environment of HERS -positive cells, and that can neutralize human HERS activation, i.e., block human HERS mediated signaling, in both the ligand-dependent and ligand-independent modes.
  • anti-human HERS antibodies that have one or more of the following characteristics: i) effectively treat cancers characterized by having one or more KRAS activating mutations, ii) demonstrate superior activity in preventing or delaying a patient's tumor from using HERS as a resistance mechanism, iii) demonstrate superior activity in preventing or delaying a patient's tumor from developing resistance to other therapeutic agents or treatment regimens such as erlotinib, cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, naveibine, eribulin, paclitaxel protein- bound particles for injectable suspension, ixabepilone, capecitabine, ramucirumab, FOLFOX (leucovonn, fluorouracil, and oxaliplatin), FOLFIRJ (leucovorin, fluorouracil, and irinotecan), pertuzumab
  • ty for purposes of improving manufacturability (e.g., ease and quality of purification, thermal and chemical stability, solubility, viscosity, and/or homogeneity), formulation stability (e.g., mitigate aggregation and self-association of the purified antibody), and/or pharmacokinetic characteristics (e.g., reduce clearance resulting from non-specific binding in vivo), while retaining acceptable binding affinity to human HER3 ECD, and/or v) otherwise, demonstrate in vivo stability, physical and chemical stability including, but not limited to, thermal stability, solubility, low self- association, and pharmacokinetic characteristics which are acceptable for development and/or use in the treatment of cancer.
  • manufacturability e.g., ease and quality of purification, thermal and chemical stability, solubility, viscosity, and/or homogeneity
  • formulation stability e.g., mitigate aggregation and self-association of the purified antibody
  • pharmacokinetic characteristics e.g., reduce clearance resulting from
  • an embodiment of the present invention provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HC VR and the LCVR together form an antigen binding site that binds to human HERS, wherein the antigen binding site comprises:
  • HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8)
  • HCDR2 comprises the amino acid sequence
  • the HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and b. three CDRs in the LCVR, wherein the LCDR1 comprises the ammo acid sequence Q S S QRV YGNKNL A (SEQ ID NO: 11 ), the LCDR2 comprises the amino acid sequence YFARTLAS (SEQ ID NO: 12), and the LCDR3 comprises the ammo acid sequence QGTYYGTNWYVG (SEQ ID NO:
  • Another embodiment of the present invention provides an antibody, or an antigen- binding fragment thereof, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a HCVR and the LC comprises a LCVR, wherein the HCVR and the LCVR together form an antigen binding site that binds to human HERS, wherein the antigen binding site comprises:
  • the HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8)
  • the HCDR2 comprises the ammo acid sequence 11 YVTYNTYY ANW A G (SEQ ID NO: 9)
  • the HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10)
  • the LCDR1 comprises the ammo acid sequence Q S S QRV YGNKNL A (SEQ ID NO: 11 )
  • the LCDR2 compnses the amino acid sequence YFARTLAS (SEQ ID NO: 12)
  • the LCDR3 comprises the ammo acid sequence QGTYYGTNWYVG (SEQ ID NO: 13).
  • the present invention provides an antibody, or an antigen-binding fragment thereof, comprising a HC and a LC, wherein the HC comprises a HCVR and the LC comprises a LCVR, wherein the HCVR has the amino acid sequence given in SEQ ID NO: 1, and the LCVR has the amino acid sequence given in SEQ ID NO: 3, and wherein the HCVR and the LCVR together form an antigen binding site that binds to human HER3.
  • the present invention provides an antibody, or an antigen-binding fragment thereof, that binds human HER3, comprising a HC and a LC, wherein the HC has the amino acid sequence given in SEQ ID NO: 2, and the LC has the amino acid sequence given in SEQ ID NO: 4.
  • the present invention provides an antibody that binds human HER3, comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence given in SEQ ID NO: 2, and each light chain has the amino acid sequence given in SEQ ID NO: 4.
  • the present invention provides an antibody that binds human HER3, comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence given in SEQ ID NO: 2, and each light chain has the amino acid sequence given in SEQ ID NO: 4, wherein one of the heavy chains forms an inter-chain disulfide bond with one of the light chains, and the other heavy chain forms an inter-chain disulfide bond with the other light chain, and one of the heavy chains forms two inter-chain disulfide bonds with the other heavy chain.
  • the antibody that binds human HER3 is glycosylated.
  • the present invention provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and the LCVR together form an antigen binding site that binds to human HER3, wherein the antigen binding site comprises:
  • the HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8)
  • the HCDR2 comprises the amino acid sequence
  • the HCDR3 comprises the ammo acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and three CDRs in the LCVR, wherein the LCDR1 comprises the amino acid sequence Q S S QRV YGNKNL A (SEQ ID NO: 11 ), the LCDR2 comprises the amino acid sequence YFARTLAS (SEQ ID NO: 12), and the LCDR3 comprises the amino acid sequence QGTYYGTNWYVG (SEQ ID NO: 13), wherein the antibody, or the antigen-binding fragment thereof, has a dissociation equilibrium constant, KD, of less than about 10 nM, of less than 5 nm, or between 5 nM and 1.0 nM for human, cyno, and mouse HER3 ECD at 37°C.
  • KD dissociation equilibrium constant
  • the present invention provides an antibody of the present invention that has a dissociation equilibrium constant, K D , of about 1.6 nM affinity at 37°C for the human HER3 ECD polypeptide as shown in SEQ ID NO: 23.
  • the antibodies of the present invention are further characterized by a k 0il rate to the human HER3 ECD as shown in SEQ ID NO: 23 of about 2,8 x 10 s M " ⁇
  • the antibody of the present invention is further characterized by a k ot - f rate to the human HER3 ECD polypeptide as shown in SEQ ID NO: 23 of about 4.4 x lO "4 sec " ' at 37°C.
  • the K D , k on , and k o ff values are established by binding kinetics at 37°C as described in "Binding Kinetics, Affinity, and Ligand Binding Potentiation Assays" below.
  • the antibodies of the present invention bind to human HER3 ECD polypeptide as shown in SEQ ID NO: 23 with high affinity.
  • the term "high affinity” refers to a K D of less than about 10 nM for the human HER3 ECD polypeptide, including, but not limited to, the HER3 FXD polypeptide as shown in SEQ ID NO: 23.
  • Certain antibodies of the present invention bind human HERS ECD with a high affinity and upon binding to HERS ECD do not block the HERS ligand NRG1 binding to HERS ECD but instead potentiate increased binding of NRG1 to HERS ECD.
  • certain antibodies of the present invention demonstrate a positive response in tumor xenograft models, such as FaDu and A253 for head and neck cancer, A549 for lung cancer, and BxPC-3 for pancreatic cancer, indicating potential as a cancer therapeutic.
  • the present invention provides a mammalian ceil, comprising a DNA molecule comprising: a) a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, and b) a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, wherein the cell is capable of expressing an antibody comprising i) a heavy chain having the ammo acid sequence of SEQ ID NO: 2, and ii) a light chain having the amino acid sequence of SEQ ID NO: 4.
  • the present invention provides a process for producing an antibody, comprising: a) a heavy chain having the amino acid sequence of SEQ ID NO: 2, and b) a light chain having the amino acid sequence of SEQ ID NO: 4, the process comprising cultivating a mammalian cell of the present invention under conditions such that the antibody is expressed, and recovering the expressed antibody.
  • the present invention provides an antibody produced by such a process.
  • the present invention provides a pharmaceutical composition, comprising an antibody of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the present in v ention pro vides methods of treating cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention.
  • the present invention provides methods of treating cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention, wherein the cancer is gastric cancer, head and neck cancer, preferably, head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, renal cancer, breast cancer, lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, colorectal cancer, or hepatocellular carcinoma (HCC).
  • HNSCC head and neck cancer
  • pancreatic cancer renal cancer
  • breast cancer lung cancer
  • lung cancer preferably, NSCLC
  • sq-NSCLC colorectal cancer
  • HCC hepatocellular carcinoma
  • such methods of treating cancer comprise the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti -neoplastic agents selected from the group consisting of cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbme, eribulin, paclitaxel protein- bound particles for injectable suspension, ixabepilone, capecitabine, ramucirumab,
  • FOLFOX (leucovoriii, fluorouraciL and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and innotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291 , ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • such methods of treating cancer comprise the administration of an effective amount of the compound of the present invention in simultaneous, separate, or sequential combination with one or more immuno-oncology agents selected from the group consisting of nivolumab, ipilimumab, pidilizumab, penibrolizumab, and durvalumab.
  • the present invention provides methods of treating breast cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present inventi on.
  • the present invention provides methods of treating breast cancer which comprise the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulm, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapaiimb, rociletinib, AZD9291, ASP8273, HM61713, and erlotinib, or
  • the present invention provides methods of treating head and neck cancer, preferably, HNSCC, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention.
  • the present invention provides methods of treating head and neck cancer, preferably, HNSCC, which comprise the administration present invention in
  • anti -neoplastic agents selected from the group consisting of ramucirumab, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletimb, AZD9291, ASP8273, ⁇ 6 ⁇ 713, and eriotinib, or a pharmaceutically acceptable salt thereof.
  • anti -neoplastic agents selected from the group consisting of ramucirumab, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel protein-bound particles for injectable suspension, ixabep
  • the present invention provides methods of treating ovarian cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention.
  • these methods of treating ovaria cancer comprise the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, cisplatin, carboplatin, liposomal doxorubicin, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713, and eriotinib, or a pharmaceutically acceptable salt thereof.
  • the present invention provides methods of treating gastric cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention.
  • these methods of treating gastric cancer comprise the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713, and eriotinib, or a
  • anti-neoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib
  • the present invention provides methods of treating HCC, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention.
  • these methods of treating HCC comprise the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more antineoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713, and erlotinib, or a
  • the present invention provides methods of treating colorectal cancer, comprising administering to a patient in need thereof, an effective amount of an antibody of the present invention.
  • these methods of treating colorectal cancer comprise the administration of an effective amount of the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of
  • ramucirumab FOLFOX (leucovorin, fluorouracil, and oxaliplatm), FOLFIRJ (leucovorin, fluorouracil, and irinotecan pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • the present invention provides an antibody of the present invention, for use in therapy.
  • the present invention provides an antibody of the present invention, for use in the treatment of cancer.
  • the present invention provides an antibody of the present invention, for use in the treatment of cancer, wherein the cancer is lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides an antibody of the present invention for use in the treatment of a KRAS wild- type cancer.
  • the KRAS wild-type cancer is lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides an antibody of the present invention for use in the treatment of a cancer containing a KRAS activating mutation.
  • the cancer containing a KRAS activating mutation is lung cancer, preferably , NSCLC, and, more preferably , sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides an antibody of the present invention in simultaneous, separate, or sequential combination wiih one or more anti-neoplastic agents selected from the group consisting of cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navel bine, eribuiin, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, ramucirumab, FOLFOX (leucovonn, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatmib, rociletinib, AZD9291,
  • the present invention provides an antibody of th e present invention in simultaneous, separate, or sequential combination with one or more immuiio- oncology agents selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, and durvalumab, for use in the treatment of cancer.
  • the present invention provides an antibody of the present invention, for use in the treatment of breast cancer.
  • the present invention provides an antibody of the present invention, for use in the treatment of KRAS wild-type breast cancer.
  • the present invention provides an antibody of the present invention for use in the treatment of breast cancer, wherein the breast cancer contains a KRAS activating mutation.
  • the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribuiin, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatmib, rociletinib, AZD9291, ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • one or more anti-neoplastic agents selected from the group consisting of ramucirumab, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribu
  • the present invention provides an antibody of the present invention, for use in the treatment of ovarian cancer. In another embodiment, the present invention provides an antibody of the present invention, for use in the treatment of KRAS wild-type ovarian cancer. In another embodiment, the present invention provides an antibody of the present invention for use in the treatment of ovarian cancer, wherein the ovarian cancer contains a KRAS activating mutation.
  • the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti -neoplastic agents selected from the group consisting of ramucirumab, cisplatin, carboplatin, liposomal doxorubicin, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • one or more anti -neoplastic agents selected from the group consisting of ramucirumab, cisplatin, carboplatin, liposomal doxorubicin, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, a
  • the present invention provides an antibody of the present invention, for use in the treatment of gastric cancer.
  • the present invention provides an antibody of the present invention, for use in the treatment of KRAS wild-type gastric cancer.
  • the present invention provides an antibody of the present invention for use in the treatment of gastric cancer, wherein the gastric cancer contains a KRAS activating mutation.
  • the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab,
  • panitumumab necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib,
  • AZD9291 ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • the present invention provides an antibody of the present invention, for use in the treatment of head and neck cancer, preferably, H SCC.
  • the present invention provides an antibody of the present in vention, for use in the treatment of KRAS wild-type head and neck cancer, preferably, HNSCC.
  • the present invention provides an antibody of the present invention for use in the treatment of head and neck cancer, preferably, HNSCC, wherein the head and neck cancer, preferably, HNSCC, contains a KRAS activating mutation.
  • the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291 , ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • one or more anti-neoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291 , ASP8273, HM61713, and
  • the present invention provides an antibody of the present invention, for use in the treatment of hepatocell ular carcinoma (HCC).
  • HCC hepatocell ular carcinoma
  • the present invention provides an antibody of the present invention, for use in the treatment of KRAS wild-type HCC.
  • the present invention provides an antibody of the present invention for use in the treatment of HCC, wherein the HCC contains a KRAS activating mutation.
  • the antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of ramucirumab, pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitmib, afatinib, neratinib, lapatinib, rociletimb,
  • AZD9291 ASP8273, HM61713, and erlotinib, or a pharmaceutically acceptable salt thereof.
  • the present invention provides an antibody of the present invention, for use in the treatment of colorectal cancer.
  • the present invention provides an antibody of the present invention, for use in the treatment of KRAS wild-type colorectal cancer.
  • the present invention provides an antibody of the present invention for use in the treatment of colorectal cancer, wherein the colorectal cancer contains a KRAS activating mutation.
  • the antibody of the present invention in simultaneous, separate, or sequential combination with one or more antineoplastic agents selected from the group consisting of ramucirumab, FOLFOX
  • the present invention provides the use of an antibody of the present invention for the manufacture of a medicament for the treatment of cancer.
  • the present invention provides the use of an antibody of the present invention for the manufacture of a medicament for the treatment of cancer, wherein the cancer is lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, H SCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides for the use of an antibody of the present invention for the manufacture of a medicament for the treatment of KRAS wild-type cancer.
  • the KRAS wild-type cancer is lung cancer, preferably, NSCLC, and, more preferably, sq- NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides for the use of an antibody of the present invention for the manufacture of a medicament for the treatment of cancer, wherein the cancer contains a KRAS activating mutation and the cancer is lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides the use of an antibody of the present invention in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of cispiatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, ramucirumab, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib,
  • AZD9291 ASP8273, HM61713, and eriotimb, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer.
  • the present invention provides an antibody of the present invention, preferably, Antibody A, i) for use in therapy, ii) for use in the treatment of cancer, iii) in simultaneous, separate, or sequential combination with an anti-VEGFR2 agent and/or an anti-EGFR agent, for use in the treatment of cancer, iv) in simultaneous, separate, or sequential combination with an anti-VEGFR2 antibody (including, but not limited to, ramucirumab) and/or an anti-EGFR agent, for use in the treatment of cancer, v) in simultaneous, separate, or sequential combination with an anti-VEGFR2 agent and/or an anti-EGFR antibody (including, but not limited to, cetuximab, necitumumab, panitumumab), for use in the treatment of cancer, vi) in simultaneous, separate, or sequential combination with an anti-VEGFR2 antibody (including, but not limited to, ramucirumab) and/or an causing-EGFR antibody (including, but not limited to, cet
  • the present invention provides i)-xii) above wherein the cancer is KRAS wild-type.
  • the present invention provides i)-xii) above wherein the KRAS wild-type cancer is lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • the present invention provides i)-xii) above wherein the cancer contains a KRAS activating mutation.
  • the present invention provides i)-xii) above wherein the KRAS mutated cancer is lung cancer, preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • lung cancer preferably, NSCLC, and, more preferably, sq-NSCLC, head and neck cancer, preferably, HNSCC, pancreatic cancer, renal cancer, gastric cancer, colorectal cancer, breast cancer or HCC.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic effect). Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy. Dosing schedules, for intravenous ( .v.) or non-intravenous administration, localized or systemic, or combinations thereof, will typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours), or as indicated by the treating physician and the patient's condition.
  • An anti-VEGFR2 Ab preferably ramucirumab, is generally effective over a wide dosage range and varies based on disease state.
  • An exemplary, non- limiting range for a therapeutically effective amount of ramucirumab for dosages per three-week cycle normally fall within the range of about 6 to 10 mg/kg, preferably about 8 to about 10 mg/kg, and most preferably about 10 mg/kg.
  • dosage levels below the lower limit of the aforesaid ranges for an anti-VEGFR2 Ab, preferably ramucirumab may be more than adequate, while in other cases smaller or still larger doses may be employed with acceptable side effects. Dosing amounts and frequencies may be determined by the physicians treating the patient.
  • the present invention provides (a) an antibody of the present invention, for use in the treatment of cancer, preferably , gastric cancer, carcinoma of the gastroesophageal junction (GEJ), HCC, lung cancer, preferably, NSCLC, bladder cancer, and RCC, and, more preferably, the metastatic types thereof, in simultaneous, separate, or sequential combination with (b) ramucirumab, wherein the time period between administration of (a) and (b) is less than about 4 hours, is less than about 12 hours, is about 1 day, is about 2 days, is about 7 days, is about 14 days, is about 1 month, or is about 2 montlis, for example.
  • cancer preferably , gastric cancer, carcinoma of the gastroesophageal junction (GEJ), HCC, lung cancer, preferably, NSCLC, bladder cancer, and RCC, and, more preferably, the metastatic types thereof, in simultaneous, separate, or sequential combination with (b) ramucirumab, wherein the time period between administration of (a) and (b) is less than
  • the subject has had surgical resection prior to administration of (a) and (b), prior to administration of (a) or (b), following administration of (a) or (b), or following administration of (a) and (b).
  • (a) or (b), or both is administered system! cally, preferably via intravenous infusion.
  • an initial dose of (b) is administered at between about 200 mg/m 2 and about 400 mg/m 2 followed by weekly infusions of between about 150 mg/m 2 and about 250 mg/m 2 .
  • (b) is administered within a dosage range of about 6 to 10 mg/kg, preferably about 8 to about 10 mg/kg, and most preferably about 10 mg/kg with dosages, one time a week or once even 7 two weeks, or on Day 1 of a 21 -day cycle.
  • the use further comprises an additional distinct cancer therapy, such as, radiotherapy (preferably, (b) is initiated about one week before initiation of radiotherapy), other EGFR inhibitor therapy, hormonal therapy, or chemotherapy, including, but not limited to, administration of FOLFIRI, paciitaxei, or docetaxel.
  • Any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof.
  • anti-neoplastic agents which are presently known in the art, or being evaluated, can be grouped into a variety of classes including, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, aromatase inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, and anti-angiogenic agents.
  • alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine.
  • anti-metabolites include, but are not limited to, daunorubicin, gemcitabine, ALIMTA® and topoisomerase inhibitors include irinotecan (CPT-1 1 ), aminocamptothecin, camptothecin, DX-8951f, topotecan (topoisomerase 1), etoposide (VP- 16), and teniposide (VM-26) (topoisomerase II).
  • anti-neoplastic agents include, but are not limited to, doxorubicin and paclitaxel.
  • the source of the radiation can be either external (external beam radiation therapy-EBRT) or internal (brachytherapy-BT) to the patient being treated.
  • the dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, the type and severity tumor being treated, and the route of administration of the agent.
  • the present invention provides (a) an antibody of the present invention, for use in the treatment of KRAS wild-type colorectal cancer, preferably, the metastatic type thereof, in simultaneous, separate, or sequential combination with (b) cetuximab or necitumumab, wherein the time period between administration of (a) and (b) is less than about 4 hours, is less than about 12 hours, is about 1 day, is about 2 days, is about 7 days, is about 14 days, is about 1 month, or is about 2 months, for example.
  • the subject has had surgical resection prior to administration of (a) and (b), prior to administration of (a) or (b), following administration of (a) or (b), or following administration of (a) and (b).
  • (a) or (b), or both is administered systemically, preferably, via intravenous infusion.
  • (b) is cetuximab
  • (b) is administered at an initial dose of between about 200 mg/m 2 and about 400 mg m 2 followed by weekly infusions of between about 150 mg/m 2 and about 250 mg/m 2 .
  • (b) when (b) is necitumumab (b) is administered within a dosage range of 400 to 1000 mg with dosages on 2 or 3 days of a 21 -day cycle, alternatively with dosages one time a week or once every two weeks, preferably about 400 to 1000 mg on Day 1, Day 8, and Day 15 of a 21-day cycle, more preferably about 600 to 900 mg on Day 1 and Day 8 of a 21 -day cycle, and most preferably about 800 mg on Day 1 and Day 8 of a 21-day cycle. Additional 21 -day cycles can be utilized as needed for treatment of the patient in need thereof.
  • the use further comprises an additional distinct cancer therapy, such as, radiotherapy (preferably, (b) is initated about one week before initiation of radiotherapy), other EGFR inhibitor therapy, hormonal therapy, or chemotherapy, including FOLFIRJ or irinotecan-based chemotherapy.
  • radiotherapy preferably, (b) is initated about one week before initiation of radiotherapy
  • other EGFR inhibitor therapy preferably, (b) is initated about one week before initiation of radiotherapy
  • hormonal therapy preferably, or a chemotherapy, including FOLFIRJ or irinotecan-based chemotherapy.
  • Any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof.
  • anti-neoplastic agents which are presently known in the art, or being evaluated, can be grouped into a variety of classes including, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, a ti -survival agents, biological response modifiers, anti- hormones, and anti-angiogenic agents.
  • alkylating agents include, but are not limited to, cisplatm, cyclophosphamide, melphaian, and dacarbazine.
  • anti-metabolites include, but are not limited to, daunorubicin, gemcitabine, ALIMTA® and topoisomerase inhibitors include irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecaii (topoisomerase 1), etoposide (VP- 16), and teniposide (VM-26) (topoisomerase II).
  • anti-neoplastic agents include, but are not limited to, doxorubicin and paclitaxel.
  • the source of the radiation can be either external (external beam radiation therapy-EBRT) or internal (brachy therapy -BT) to the patient being treated.
  • the dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, the type and severity tumor being treated, and the route of administration of the agent.
  • the present invention provides (a) an antibody of the present invention, preferably, Antibody A, for use in the treatment of head and neck cancer, preferably, HNSCC, in simultaneous, separate, or sequential combination with (b) cetuximab or necitumumab, wherein the time period between administration of (a) and (b) is less than about 4 hours, is less than about 12 hours, is about 1 day, is about 2 days, is about 7 days, is about 14 days, is about 1 month, or is about 2 months, for example.
  • (a), or (b), or both is administered systemically, preferably via intravenous infusion.
  • (b) when (b) is cetuximab, (b) is administered at a an initial dose of between about 200 mg/m 2 and about 400 mg/n followed by weekly infusions of between about 150 mg/m 2 and about 250 mg/m .
  • (b) when (b) is necitumumab (b) is administered within a dosage range of 400 to 1000 nig with dosages on 2 or 3 days of a 21 -day cycle, alternatively with dosages one time a week or once every two weeks, preferably about 400 to 1000 mg on Day 1, Day 8, and Day 15 of a 21-day cycle, more preferably about 600 to 900 mg on Day 1 and Day 8 of a 21 -day cycle, and most preferably about 800 mg on Day 1 and Day 8 of a 21-day cycle.
  • the use further comprises an additional distinct cancer therapy, such as, radiotherapy (preferably, (b) is initated about one week before initiation of radiotherapy), other EGFR inhibitor therapy, hormonal therapy, or chemotherapy, including platinum- based chemotherapy (e.g., cisplatin, oxaliplatin, or FOLFOX).
  • radiotherapy preferably, (b) is initated about one week before initiation of radiotherapy
  • other EGFR inhibitor therapy preferably, (b) is initated about one week before initiation of radiotherapy
  • hormonal therapy e.g., adaliplatin, or FOLFOX.
  • platinum- based chemotherapy e.g., cisplatin, oxaliplatin, or FOLFOX
  • the present invention provides (a) an antibody of the present invention, preferably, Antibody A, for use in the treatment of NSCLC, preferably, squamous cell NSCLC (sq-NSCLC), in simultaneous, separate, or sequential combination with (b) cetuximab or necitumumab, wherein the time period between administration of (a) and (b) is less than about 4 hours, is less than about 12 hours, is about 1 day, is about 2 days, is about 7 days, is about 14 days, is about 1 month, or is about 2 months, for example.
  • (a) or (b), or both are administered systemically, preferably, via intravenous infusion.
  • (b) is cetuximab (b) is administered at an initial dose of between about 200 mg/m 2 and about 400 mg/m 2 followed by weekly infusions of between about 150 mg/m 2 and about 2.50 mg/m 2 .
  • (b) when (b) is necitumumab (b) is administered within a dosage range of 400 to 1000 mg with dosages on 2 or 3 days of a 21-day cycle, alternatively with dosages one time a week or once every two weeks, preferably about 400 to 1000 rng on Day I, Day 8, and Day 15 of a 21 -day cycle, more preferably about 600 to 900 mg on Day 1 and Day 8 of a 21 -day cycle, and most preferably about 800 mg on Day 1 and Day 8 of a 21 -day cycle. Additional 21 -day cycles can be utilized as needed for treatment of the patient in need thereof.
  • the use further comprises an additional distinct cancer therapy, such as, radiotherapy (preferably, (b) is initiated about one week before initiation of radiotherapy), other EGFR inhibitor therapy, hormonal therapy, or chemotherapy, including platinum-based chemotherapy (e.g., cisplatin, oxaliplatin, or FOLFOX) and/or anti-metabolite chemotherapy (e.g., gemcitabine).
  • radiotherapy preferably, (b) is initiated about one week before initiation of radiotherapy
  • other EGFR inhibitor therapy e.g., hormonal therapy, or chemotherapy, including platinum-based chemotherapy (e.g., cisplatin, oxaliplatin, or FOLFOX) and/or anti-metabolite chemotherapy (e.g., gemcitabine).
  • platinum-based chemotherapy e.g., cisplatin, oxaliplatin, or FOLFOX
  • anti-metabolite chemotherapy e.g., gemcitabine
  • gemcitabine anti-metabolite chemotherapy
  • (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) ca be administered at the same time as each administration of (b). Where (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) can be administered subsequent to each administration of (b). Where (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) can be administered prior to, at the same time as, or subsequent to, each administration of (b) or some combination thereof. Where (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) can be administered at different intervals in relation to therapy with (b). Where (b) is administered at repeated intervals (e.g.
  • (a) can be administered in a single or series of dose(s) prior to, at any time during, or subsequent to the course of treatment with (b).
  • (b) is administered at repeated intervals (e.g. during a standard course of treatment)
  • (a) can be administered in a single dose prior to, at any time during, or subsequent to the course of treatment with (b).
  • (b) is administered at repeated intervals (e.g. during a standard course of treatment)
  • (a) can be administered in a single dose prior to the course of treatment with (b).
  • (b) is administered at repeated intervals (e.g. during a standard course of treatment)
  • (a) can be administered in a single dose at any time during the course of treatment with (b).
  • (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) can be administered in a single dose subsequent to the course of treatment with (b). Where (b) is administered at repeated interval s (e.g. during a standard course of treatment), (a) can be administered in a series of doses prior to the course of treatment with (b). Where (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) can be administered in a series of doses subsequent to the course of treatment with (b). Where (b) is administered at repeated intervals (e.g. during a standard course of treatment), (a) can be administered in a series of doses subsequent to the course of treatment with (b).
  • the present invention provides the use of an antibody of the present invention, preferably, Antibody A, in simultaneous, separate, or sequential combination with one or more anti-neoplastic agents selected from the group consisting of cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, naveibme, eribulin, paclitaxel protein-bound particles for injectable suspension, ixabepilone, capecitabine, ramucirumab, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, necitumumab, gefitinib, afatinib, neratinib, lapatmib, rocile
  • an antibody of the present invention is an engineered, non-naturally occurring polypeptide complex. Unless indicated otherwise, the term “antibody” refers to an immunoglobulin molecule comprising two heavy chains and two light chains
  • each chain includes a variable region of about 110 to about 120 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein.
  • CDRs complementarity determining regions
  • the carbox -terminal portion of each chain defines a constant region primarily responsible for effector function.
  • an antigen-binding fragment refers to any antibody fragment that retains the ability to bind to its antigen.
  • Such "antigen -binding fragments” can be selected from the group consisting of Fv, scFv, Fab, F(ab Fab', scFv-Fc fragments and diabodies.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable region.
  • an antigen-binding fragment comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).
  • an antigen-binding fragment as used herein comprises a HCVR and a LCVR which confers antigen-binding specificity to human HER3 (i.e., a "human HER 3 binding fragment") or human HER3-ECD (i.e., a "human HER3-ECD binding fragment”).
  • LCVR light chain variable region
  • LCVR refers to a portion of a LC of an antibody molecule that includes ammo acid sequences of Complementarity Determining Regions (CDRs; i.e., LCDR1 , LCDR2, and LCDR3), and Framework Regions (FRs).
  • CDRs Complementarity Determining Regions
  • FRs Framework Regions
  • HCVR ' heavy chain variable region
  • HCVR refers to a portion of a HC of an antibody molecule that includes amino acid sequences of Complementarity Determining Regions (CDRs; i.e., HCDRl, HCDR2, and HCDR3), and Framework Regions (FRs).
  • An antibody of the present invention is desi gned to have engineered CDRs and have some portions of the antibody (all or parts of the frameworks, hinge regions, and constant regions) to be of human origin that are identical with or substantially identical (substantially human) with frameworks and constant regions derived from human genomic sequences.
  • Fully human frameworks, hinge regions, and constant regions are those human germline sequences as well as sequences with naturally-occurring somatic mutations and those with engineered mutations.
  • An antibody of the present invention may comprise framework, hinge, or constant regions derived from a fully huma framework, hinge, or constant region containing one or more amino acid substitutions, deletions, or additions therein. Further, an antibody of the present invention is preferably substantially non-immunogenic in humans.
  • the antibody of the present invention is an IgG type antibody and has four amino acid chains (two "heavy” chains and two ' " light” chains) that are cross-linked via intra- and mter-chain disulfide bonds.
  • Each heavy chain is comprised of an N-terminal HCVR and a heavy chain constant region ("HCCR").
  • Each light chain is comprised of a LCVR and a light chain constant region (' " LCCR”).
  • antibodies having native human Fc sequences are glycosylated in the Fc region. Typically, glycosylation occurs in the Fc region of the antibody at a highly conserved N- glycosylation site. N-glycans typically attach to asparagine.
  • Antibodies may be glycosylated at other positions as well.
  • the antibody of the present invention contains an Fc portion which is derived from human IgG] Fc region. Furthermore, in some embodiments, the antibody of the present invention contains an Fc portion which is derived from human IgG] Fc region. Furthermore, in some embodiments, the antibody of the present invention contains an Fc portion which is derived from human IgG] Fc region. Furthermore, in some embodiments, the antibody of the present invention contains an Fc portion which is derived from human IgG] Fc region.
  • antibodies of the present invention contain an IgG] heavy chain with a C- terminal lysine deletion.
  • CDRs complementarity determining regions
  • the terms ''complementarity determining region" and “CDR” refer to the non-contiguous antigen combining sites found within the variable region of LC and HC polypeptides of an antibody or an antigen-binding fragment thereof. These particular regions have been described by others including Kabat, et a!., Ann, NY Acad, Sci. 190:382-93 (1971); abat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, et al, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.
  • Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 the three CDRs of the heavy chain are referred to as "HCDRl, HCDR2, and HCDR3"
  • the three CD Rs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3".
  • the CDRs contain most of the residues which form specific interactions with the antigen.
  • the Kabat CDR definition (Kabat et al., " 'Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)) is based upon antibody sequence variability.
  • the Chothia CDR definition (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., ''Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) is based on three-dimensional structures of antibodies and topologies of the CDR l oops.
  • the Chothia CDR definitions are identical to the Kabat CDR definitions with the exception of HCDRl and HCDR2.
  • the North CDR definition (North et al., "A New Clustering of Antibody CDR Loop
  • HCDR1 and HCDR3 also use the North definition.
  • HCDR2 uses a hybrid of North and Kabat definitions. The North definition is used to identify the starting N-terminal site while Kabat is used to define the last position.
  • HCDR3, TRWGDONGLDI SEQ ID NO: 10
  • a DNA molecule of the present invention is a non-naturally occurring DNA molecule that comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence of at least a variable region polypeptide of an antibody of the present invention.
  • An isolated DNA encoding a HCVR region can be converted to a full- length heavy chain gene by operably linking the HCVR-encoding DNA to another DNA molecule encoding heavy chain constant regions.
  • the sequences of human, as well as other mammalian, heavy chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained e.g., by standard PCR
  • An isolated DNA encoding a LCVR region may be converted to a full-length light chain gene by operably linking the LCVR-encoding DNA to another DNA molecule encoding a light chain constant region.
  • the sequences of human, as well as other mammalian, light chain constant region genes are known in the art. DNA fragments encompassing ihese regions can he obtained by standard PGR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • the polynucleotides of the present invention may be expressed in a host cell after the sequences have been operably linked to an expression control sequence.
  • the expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences.
  • the antibody of the present invention may readily be produced in mammalian cells such as CHO, NS0, HEK293 or COS ceils.
  • the host cells may be cultured using techniques well known in the ait.
  • the vectors containing the polynucleotide sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host.
  • the antibody, or the nucleic acids encoding the same is provided in isolated form.
  • isolated refers to a protein, peptide, or nucleic acid which is free or substantially free from any other macromolecular species found in a cellular environment.
  • substantially free as used herein means the protein, peptide, or nucleic acid of interest comprises between about 80% and about 99% (on a molar basis) of the macromolecular species present, preferably, between about 90% and about 99%, and, more preferably, between about 95% and about 99%.
  • the antibody of the present in v ention, or pharmaceutical compositions comprising the same may be administered by parenteral routes (e.g., subcutaneous and intravenous).
  • An antibody of the present invention may be administered to a patient alone with pharmaceutically acceptable carriers, diluents, or excipients in single or multiple doses.
  • Pharmaceutical compositions of the present invention can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 19 th ed.
  • the term "patient” refers to a mammal, preferably a human.
  • cancer * ' and "cancerous” refer to or describe the physiological condition in patients that is typically characterized by unregulated cell proliferation. Included in this definition are benign and malignant cancers.
  • head stage cancer or “early stage tumor” is meant a cancer that is not advanced or metastatic or is classified as a Stage 0, 1, or II cancer.
  • Examples of cancer include, but are not limited to, gastric, lung, including, NSCLC and sq-NSCLC, head and neck, including, HNSCC, pancreatic, renal, breast, or colorectal cancer, or HCC.
  • treating refers to restraining, slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • k is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen or antibody fragment-antigen interaction.
  • binding as used herein in reference to the affinity of an antibody for human HER3 is intended to mean, unless indicated otherwise, a K D of less than about 1 x 10 "8 M, preferably, less than about 5 x lO "9 M, more preferably, less than about 1 x 10 "9 M as determined by common methods known in the art, including by use of a surface plasmon resonance (SPR) biosensor at 25°C or 37°C essentially as described herein.
  • SPR surface plasmon resonance
  • HER3 selective or “selectivity" ' used herein in reference to a compound of the present invention refers to a compound that binds a target, such as human HER3 and/or human HER3 ECD, with a D about 1000-, about 500-, about 200-, about 100-, about 50-, or about 10-fold lower than the compound binds other proteins, including member of the HER/EGFR family such as human HER2, as measured by surface plasmon resonance at 25°C or 37°C.
  • an HER3 selective aniibody of the present invention binds human HERS but does not bind or only minimally binds human HER2 when assayed by the methods described in the Example herein below.
  • HER3 HER3 polypeptide
  • ErbB3 ErhB3 polypeptide
  • ErhB3 polypeptide are used interchangeably herein and, unless otherwise indicated, are intended to refer to the mature form (i.e., does not include the signal peptide) of the human receptor tyrosine- protem kinase erhB-3, as well as mutated, and/or truncated forms thereof, that bind human neuregulin- 1.
  • HER3 include, e.g., a human polypeptide encoded by the nucleotide sequence provided in CBI GenBank accession number M34309, the human HER3 protein encoded by the polypeptide sequence provided in NCBI GenPept accession number AAA35979, and/or the polypeptide having the amino sequence shown in, for example, SEQ ID NO: 16.
  • Nucleotide sequences encoding murine, rhesus monkey, and cynomolgus monkey HER3 proteins are provided in NCBI GenBank accession numbers AY686636.1, AFI35044.1, and EHH66388.1, respectively.
  • HER3 proteins are frequently generated lacking their signal peptides and/or fused, typically at the C-terminus of the HER3 protein, to one or more other peptides, in order to aid in purification and/or other laboratory
  • human HER3 BCD' * refers to HER3 proteins with or without a signal peptide including, but not limited to, one or more of the human HER3 ECD proteins as shown in SEQ ID NOs: 7, 21, 22, or 23.
  • the phrase "effective amount” means the amount of an antibody of the present invention or pharmaceutical composition comprising an antibody of the present invention that will elicit the biological or medical response of or desired therapeutic effect on a tissue, system, animal, mammal or human that is being sought by the researcher, medical doctor, or other clinician.
  • An effective amount of the antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual .
  • An effective amount is also one in which any toxic or detrimental effect of the antibody is outweighed by the therapeutically beneficial effects.
  • an effective amount can be readily determined by the attending medical doctor, diagnostician, or other clinician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
  • determining the effective amount for a patient a number of factors are considered by the attending diagnostician, including, but not limited to: the species of patient; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
  • the phrase “in combination with” refers to the administration of an antibody of the present invention or a pharmaceutically acceptabl e formul ation thereof, with another therapeutic agent simultaneously.
  • the phrase “in combination with” also refers to the administration of an antibody of the present invention or a pharmaceutically acceptable formulation thereof, with another therapeutic agent sequentially in any order.
  • the phrase “in combination with” also refers to the administration of an antibody of the present invention or a pharmaceutically acceptable formulation thereof, with another therapeutic agent in any combination thereof.
  • An antibody of the present invention, or a pharmaceutically acceptable formulation thereof can be administered prior to administration of another therapeutic agent.
  • An antibody of the present invention, or a pharmaceutically acceptable formulation thereof can be administered at the same time as administration of another therapeutic agent.
  • An antibody of the present invention, or a pharmaceutically acceptable formulation thereof can be administered subsequent to administration of another therapeutic agent.
  • An antibody of the present invention, or a pharmaceutically acceptable formulation thereof can be administered prior to, at the same time as, or subsequent to administration of another therapeutic agent, or in some combination thereof.
  • responsiveness to treatment with an antibody of the present invention, or “therapeutic effect” refers to the clinical or therapeutic benefit(s) imparted to a patient upon administration of i) an antibody of the present invention, preferably.
  • Antibody A in combination with an ami- VEGFR2 agent and/or an anti-EGFR agent, iii) an antibody of the present invention, preferably, Antibody A, in combination with an anti-VEGFR2 antibody and/or an ami- EGFR agent, iv) an antibody of the present invention, preferably, Antibody A, in combination with an anti-VEGFR2 agent and/or an anti-EGFR antibody, v) an antibody of the present invention, preferably, Antibody A, in combination with an anti-VEGFR2 antibody and/or an anti-EGFR antibody, vi) an antibody of the present invention, preferably, Antibody A, in combination with ramucirumab, necitumumab, and/or cetuximab, vii) an antibody of the present invention, preferably, Antibody A, in combination with ramucirumab and cetuximab, viii) an antibody of the present invention, preferably, Antibody A, in combination with ramucirumab and necitumumab,
  • the manner of th e invention is such that therapy with a combination described herein is synergistic in nature, although in alternative embodiments the combination provides additive benefits for therapy.
  • benefit(s) include any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response
  • tumor regression including a complete response or a partial response
  • tumor weight or size shrinkage longer time to disease progression, increased duration of survival, longer progression free survival, improved overall response rate, increased duration of response, and improved quality of life and/or improving signs or symptoms of cancer, etc.
  • An unexpected therapeutic effect of the combination treatments of the invention is the ability to produce marked anti-cancer effects in a patient without causing significant toxicities or adverse effects, so that the patient benefits from the combination treatment method overall.
  • the efficacy, i.e., therapeutic effect(s), of the combination treatment of the invention can be measured by various endpoints commonly used in evaluating cancer treatments, including, but not limited to, tumor regression, tumor weight or size shrinkage, time to disease progression, overall survival, progression free survival, overall response rate, duration of response, and/or quality of life.
  • the antibodies of the present invention and combination treatments of the invention may cause inhibition of metastatic spread without shrinkage of the primary tumor, may induce shrinkage of the primary tumor, or may simply exert a tumoristatic effect.
  • novel approaches to determining efficacy, i.e., therapeutic effect(s), of any particular combination therapy of the present invention can be optionally employed, including, for example, measurement of plasma or urinary markers of angiogenesis and measurement of response through radiological imaging.
  • CR 'Complete Response
  • PR Partial Response
  • the term ''Progressive Disease refers to at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. For the avoidance of doubt, the appearance of one or more new lesions is also considered progression.
  • SD Stable Disease
  • CR refers to definitions according to RECIST v 1. 1 , Eisenhauer et al., European Journal of Cancer, 2009, 45, 228-247.
  • time to disease progression refers to the time, generally measured in weeks or months, from the time of initial treatment, until the cancer progresses or worsens. Such progression can be evaluated by the skilled clinician.
  • the term “extending TTP” refers to increasing the time to disease progression in a treated patient relative to i) an untreated patient or relative, or ii) a patient treated with less than all of the anti-neoplastic agents in a particular combination therapy.
  • the term “survival” refers to the patient remaining alive, and includes overall survival as well as progression free survival.
  • 'overall survival refers to the patient remaining alive for a defined period of time, such as 1 year, 5 years, etc., from the time of diagnosis or treatment.
  • progression free survival refers to the patient remaining alive, without the cancer progressing or getting worse.
  • the term "extending survival" is meant increasing overall or progression free survival in a treaied patieni relative to i) an untreated patient, ii) a patient treated with less than all of the anti -neoplastic agents in a particular combination therapy, or iii) a control treatment protocol. Survival is monitored for at least about one month, at least about one month, at least about two months, at least about four months, at least about six months, at least about nine months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., following the initiation of treatment or following the initial diagnosis of cancer.
  • primary tumor or “primary cancer” is meant the original cancer and not a metastatic lesion located in another tissue, organ, or location in the patient's body.
  • ramucirumab also known as Cyramza®, IMC- 1 121b, and/or CAS registry number 947687-13-0, refers to a fully human monoclonal antibody directed against human VEGFR2 comprising: two light chains, each having the amino acid sequence given in SEQ ID NO: 19, and two heavy chains, each having the amino acid sequence given in SEQ ID NO: 20.
  • Ramucirumab and methods of making and using ramucirumab including for the treatment of neoplastic diseases such as solid and non- solid tumors are disclosed in WO 2003/075840.
  • Ramucirumab (Cyramza®) is approved by the US F.D.A. as a single agent, or in combination with paclitaxel, for the treatment of patients with advanced or metastatic gastric or gastroesophageal (GE) junction adenocarcinoma with disease progression on or after prior fluoropyrimidine- or platinum-containing chemotherapy; and in combination with docetaxel, is indicated for the treatment of patients with metastatic NSCLC with disease progression on or after platinum-based chemotherapy .
  • GE gastroesophageal
  • anti-VEGFR2 Ah refers to an antibody comprising a
  • the anti-VEGFR2 Ab binds to VEGFR2 with sufficient affinity and specificity.
  • the anti-VEGFR2 Ab is an antibody comprising a light chain whose amino acid sequence is that given in SEQ ID NO 19, and a heavy chain whose amino acid sequence is that given in SEQ ID NO 20 and that binds to VEGFR2 with sufficient affinity and specificity.
  • the anti- VEGFR2 Ab is ramucirumab.
  • the antibody selected will have a sufficiently strong binding affinity for VEGFR2.
  • the antibody will generally bind VEGFR2 with a K d value of between about 100 nM - about 1 pM.
  • Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BiacoreTM assay is described in PCT Application Publication No. WO2005/012359); enzyme-linked immunoabsorbent assay (ELISA); and competition assays (e.g. a radiolabeled antigen binding assay (RIA)), for example.
  • Kd is measured by a R1A performed with an anti-VEGFR2 Ab, preferably, ramucirumab.
  • DCiOl or "LSN3180389” refers to a rat monoclonal antibody directed against mouse VEGFR2 that may be used in experiments as a surrogate in mice for an anti-VEGFR2 Ab, preferably ramucirumab. See, for example, Witte, L., et al. Cancer Metastasis Rev., 17: 155-161 (1998); Prewett, M., et al., Cancer Res. , 59: 5209-5218 (1999),
  • necitumumab refers to a recombinant IgGl human monoclonal antibody targeting the epidermal growth factor receptor (EGFR).
  • Necitumumab and methods of making and using this antibody including for the treatment of neoplastic diseases such as solid and non-solid tumors are disclosed in US 7,598,350.
  • Necitumumab is also known as IMC-1 1 F8 and/or CAS registry number 906805-06-9.
  • necitumumab has also been reported in patients with NSCLC (Thatcher, N , et al. 1 Cli Oncol 32.5 Suppl (2014) and ASCO presentation, abstract 8008, 2014 discussing "A randomized, multicenter, open-label, phase III study of gemcitabine-cisplatin (GC) chemotherapy plus necitumumab (IMC-1 1 F8/LY3012211) versus GC alone in the first-line treatment of patients (pis) with stage IV squamous non- small ceil lung cancer (sq-NSCLC)").
  • GC gemcitabine-cisplatin
  • IMC-1 1 F8/LY3012211 necitumumab
  • sq-NSCLC stage IV squamous non- small ceil lung cancer
  • cetuximab also known as Erbitux®, antibody C225, and/or IMC-C225 refers to a chimeric mouse/human anti-EGFR monoclonal antibody composed of the Fv regions of a murine anti-EGFR antibody with human IgGl heavy and kappa light chain constant regions and has an approximate molecular weight of 152 kDa.
  • Cetuximab binds specifically to the extracellular domain of the human EGFR, and is an EGFR inhibitor, which blocks ligand binding to EGFR, prevents receptor activation, and inhibits growth of tumor ceils that express EGFR. Cetuximab has been approved for use in combination with or without irinotecan in the treatment of patients with epidermal growth factor receptor-expressing, metastatic colorectal cancer who are refractory or can not tolerate irinotecan-based chemotherapy.
  • polypeptides of the variable regions of the heavy chain and light chain, the complete HC and LC amino acid sequences of anti-HER3 Antibody A (also referred to herein as, Antibody A), and the nucleotide sequences encoding the same, are listed below in the section entitled "Amino Acid and Nucleotide Sequences.”
  • SEQ ID NOs corresponding to the amino acid sequences for the LC, HC, LCVR, and HCVR of Antibody A are shown in Table 1.
  • the antibodies of the present invention including, but not limited to, Antibody A can be made and purified essentially as follows.
  • An appropriate host cell such as HEK 293 or CHO, can be either transiently or stably transfected with an expression system for secreting antibodies using an optimal predetermined HC:LC vector ratio (such as 1 :3 or 1 :2) or a single vector system encoding both the HC and the LC.
  • Clarified media into which the antibody has been secreted, may be purified using any of many commonly-used techniques.
  • the medium may be conveniently applied to a MabSeiect column (GE Healthcare), or KappaSelect column (GE Healthcare) for Fab fragment, that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4).
  • a compatible buffer such as phosphate buffered saline (pH 7.4).
  • the column may be washed to remove nonspecific binding components.
  • the bound antibody may be eluted, for example, by pH gradient (such as 20 mM Tns buffer, pH 7 to 10 mM sodium citrate buffer, pH 3.0, or phosphate buffered saline, pH 7.4, to 100 mM glycine buffer, pH 3.0).
  • Antibody fractions may be detected, such as by SDS- PAGE, and then may be pooled.
  • the antibody may be concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, multimodal, or hydroxyapatite chromatography. The purity of the antibody after these
  • chromatography steps is between about 95% to about 99%.
  • the product may be held refrigerated, immediately frozen at -70°C, or may be lyophilized.
  • SPR surface plasmon resonance
  • the binding kinetics of an anti-HER3 antibody of the invention to human, mouse and'or cyiiomoigus monkey HER3 ECDs may be determined by use of a surface plasmon resonance biosensor such as a BIAcore® 2000, BIAcore® 3000, or a BIAcore® Tl 00 (GE Health Care, Piscataway, NJ) according to methods known in the art. Except as noted, all reagents and materials may be purchased from Biacore * and measurements may be performed at 25°C or 37°C.
  • Human and mouse HER3 ECD proteins (such as those shown in SEQ ID NOs: 22, 23, and 24, respectively) which contain a C-terminal 6x histidine tag can be expressed in HEK293 cells and purified by nickel charged IMAC.
  • Rhesus mature HER3-ECD which is 100% identical to cyno mature HER3-ECD, can be purchased from Sino Biological (Beijing, P.R. China; catalog number; 90043-K08H).
  • Protein A surface for capture of antibodies may be prepared using the following methods. Soluble Protein A (Calbiochem, catalog number: 539202) may be immobilized on a CM4 (Biacore # BR- 1005-39) using manufactures recommended EDC/NHS amine coupling method (Biacore, catalog number: BR-1000-50). For example, the surfaces of all four flow cells may be cleaned by three sequential 30 second injections of a pH 1.5 glycine buffer. Then the surface may be activated by a seven minute injection of a 1 : 1 mixture of EDC/NHS.
  • soluble Protein A diluted to 50 ⁇ »/ ⁇ in 10 rnM acetate buffer, pH 4.5, may immobilized though a three minute injection across flow ceils (Fc) 1 and 2. Un-reacted sites still remaining on the chip surface may be blocked with a seven minute injection of ethanolamine.
  • Running buffer may be HBS-EP+ (Biacore # BR- 1006-69) at 25°C. Approximately 400-600RU of Protein A may be amine-coupled to the CM4 chip,
  • Antibody A may be diluted to 1 ⁇ ig/mL in running buffer. Discrete concentrations of human, mouse, and rhesus (cyno) soluble HER3-ECD ligands ranging from 125 nM to 0.24 nM can be prepared using a two-fold serial dilution into running buffer. The instrument may be equilibrated to 37°C and each kinetic cycle may consist of a series of four separate steps: (1) capture of antibody A only on Fc2, (2) injection (using kinject) of 200 ⁇ .
  • Antibody A binds to HER3-ECD as shown in SEQ ID NO: 23, Rhesus HER3-ECD (i.e., cynomolgus monkey HER3-ECD) and mouse mature HER3 ECD as shown in SEQ ID NO: 24 with an approximately similar 3 ⁇ 4 of 1.6, 2.1, and 3.0 nM, respectively (Table 2). Binding stoichiometry for all three HER3-ECD ligands were close to 2 (2 moles of ligand per one mole Antibody A) indicating that Antibody A can simultaneously bind two HER3-ECD ligands.
  • HER antibodies to inhibit or potentiate binding of biotinylated NRG1 to plate-bound HER3 ECD in an ELISA format may be determined essentially as follows.
  • Human HER3 ECD protein may be bound at 2 ⁇ ' ⁇ , to ELISA plates overnight at 4°C. Plates may be washed and then blocked with Blotto (Thermo) blocker for 1 hour at 37°C. Again plates may be washed and dose titrations of anti-HER3 antibodies may be added as 2X concentrations immediately followed by the addition of biotinylated-NRGl for a final concentration of 50 ng/mL. Plates may be incubated for 2 hours with shaking at room temperature and then washed. Streptavidin-HRP may be added to the plates and incubated for 30 minutes with shaking at room temperature. The plates may be washed and then developed with the addition of 3, 3', 5, 5'-Tetramethylbenzidine (TMB) (SurModics) followed by stop solution after 20 minutes. Plates may be read at OD 450 ilffl.
  • TMB 3, 3', 5, 5'-Tetramethylbenzidine
  • anti-HER3 Antibody A does not block binding of biotinylated-NRGl to plate-bound human HER3 ECD (as shown in SEQ ID NO: 23) but instead potentiates increased binding of biotinylated-NRGl to plate-bound human HER3 ECD.
  • HER3 antibodies 2C2, Ul-59, and Dl lf all inhibit binding of NRG 1 to human HER3 ECD.
  • MOR10703_A50V_N52S see, US 2012/0107306
  • HER3 antibody neither inhibits nor potentiates NRGl binding to human HER3 ECD.
  • the activity of anti-HER3 antibodies on HER3-HER2 heterodimerization in both ligand-dependent and ligand-independent contexts may be measured in a luciferase complementation assay essentially as follows. Briefly, 0.4 million cells per well from a HEK293 stable clone expressing HER3 tagged with N-terminal firefly luciferase and HER2 tagged with C -terminal firefly luciferase may be plated in 0.4 mL in a 24-weil plate.
  • test and control antibodies may ⁇ be added at 35 and incubated for 1 hour at 37°C followed by a 5 minute stimulation with 300 ng/mL NRGl (R&D Systems; Minneapolis, MN; catalog number 377-HB/CF) and subsequent measurement of luciferase activity for the ligand-dependent assay ( Figure 2A).
  • antibodies may be added at 35 ⁇ ' ⁇ for a 1 hour incubation at 37°C followed by measurement of luciferase activity for the ligand- independent assay ( Figure 2B). Luciferase activity was measured using the Steady-light kit (Perkm Elmer 6016751 ),
  • Antibody A ability to inhibit HER3 activation (and resulting tumor activity), especially considering that anti-HER3 Antibody A does not block NRGl binding to HERS.
  • Anti-HER3 Antibody A induced internalization and cell surface depletion of HERS The in vitro induction of internalization and depletion of cell surface HER3 by an anti-HER3 antibody may be measured in a cell based assay.
  • the aforementioned assay may be used to study the ability of antibodies of the present invention to induce clearance of HER3 in cancer cells, potentially negating HER3 mediated tumorigenic or drug resistance mechanisms in cancer cells.
  • BT474 cells express a relatively high level of ceil surface HER3 and were chosen to characterize anti-HER3 antibody induction of HER3 internalization and cell surface depletion. Briefly stated, 300,000 BT474 cells/well may be plated in each well of 6-well plates and incubated 24 hours at 37°C, 5% C0 2 . Each well may be aspirated and 1 mL of fresh growth media added containing 100 nM HER3 test antibodies. After overnight treatment at 37°C, 5% CO 2 , cells may be removed from wells with a non-enzymatic buffer. Cells are then stained 1 hour at 4°C with 1 ⁇ ug/rnl. biotinyiated 1 B4C3 HER3 antibody (BioLegend). 1 B4C3 binds to HER3 ECD but does not compete with any of the test antibodies used in this assay. Cells may be washed and then stained with
  • streptavidin-PE for 30 minutes at 4°C and then characterized by FACS analysis. Percent internalization may be determined by comparison to the untreated control. The mean and standard deviation from three experiments may be calculated and graphed.
  • anti ⁇ HER3 Antibody A induces HER3 internalization and cell surface depletion from BT474.
  • Antibody A internalizes HER3 similar to, or better than, certain other HER3 antibodies known in the art including MOR10703_A50V_N52S, Ul -59, and Ab#6 (see, US
  • test antibodies alone and in combination with other agents may be may be measured in cancer xenograft models such as A549 (lung; KRAS mutation), SCCHN A- 253 (head and neck tumor type), FaDu (head and neck tumor type), BxPC-3 (pancreatic tumor type) utilizing approved animal research methods that are compliant with international, national, and local laws and guidance for example those approved by the Institutional Animal Care and Use Committee and performed in accordance with current regulations and standards of the United States Department of Agriculture and the National Institute of Health.
  • cancer xenograft models such as A549 (lung; KRAS mutation), SCCHN A- 253 (head and neck tumor type), FaDu (head and neck tumor type), BxPC-3 (pancreatic tumor type) utilizing approved animal research methods that are compliant with international, national, and local laws and guidance for example those approved by the Institutional Animal Care and Use Committee and performed in accordance with current regulations and standards of the United States Department of Agriculture and the National Institute of Health.
  • mice bearing xenograft tumors at approximately 300 mm' volume, randomized at 10 mice/group by tumor size and bod ⁇ ' - weight by randomization techniques well known in the art, may be treated with control IgG or test antibodies, or combinations of test antibodies.
  • the duration of treatment may be determined by: 1 ) a study endpoint being reached (i.e. , statistically significant inhibition of tumor growth is achieved, or no anti-tumor effect is apparent), or 2) a clinical endpomt being reached (e.g. , tumor burden is impacting animal welfare or survival).
  • Control IgG, anti-HER3 Antibody A, cetuximab, or necitumumab may be formulated in phosphate buffered saline.
  • PBS formulated anti-HER3 Antibody A or control isotype IgG may be dosed at 20 mg/kg IP twice a week for 4 weeks.
  • PBS formulated anti-EGFR antibodies, cetuximab or necitumumab for example, may be dosed by intravenous (i.v.) administration at 20 mg/kg once every week.
  • anti-HER3 Antibody A and human monoclonal anti-EGFR antibody cetuximab or necitumumab, as well as their respective monotherapies may be tested in xenograft models across multiple tumor types (for example, squamous NSCLC, squamous bladder, squamous head and neck, squamous thyroid, non- squamous lung large cell, squamous anal or non-squamous colorectal cancer with and without activating mutations (e.g., RAS mutations)).
  • Antitumor efficacy of the treatment groups may be assessed by measuring tumor volume via caliper measurements twice a week during the course of the study.
  • Body weight may be measured regularly, twice weekly, for example, during the course of the study as a general indicator of toierability. Differences between treatments may be considered statistically significant if p ⁇ 0.05.
  • the antitumor efficacy of the experimental treatments may be expressed as the T/C ratio (in percent) and may be calculated as summarized below.
  • ⁇ mean tumor volume of the drug-treated group on the final day of the study minus mean tumor volume of the drug-treated group on initial day of dosing
  • AC mean tumor volume of the control group (specified in each study ) on the final day of the study minus mea tumor volume of the control group on initial day of dosing.
  • Tumor volume data may be analyzed with a two-way repeated measures analysis of variance by time and treatment using the MIXED procedures in SAS software (Version 9.3).
  • the response analyzed is the log transformation of tumor volume as necessary to equalize the variance across time and treatment groups.
  • Spatial Power may be used for the correlation structure for the repeated measures model. Predefined pairwise comparisons of treated group(s) to control group(s) may be conducted for each time point.
  • Mean tumor volume data may be expressed as the geometric mean + sem.
  • anti-HER3 Antibody A monotherapy treatment groups exhibited a statistically significant reduction in tumor volume when compared to control IgG in xenograft models SCCHN A-253 (head and neck tumor type), A549 (lung tumor containing KRAS mutation), FaDu (head and neck tumor type), and BxPC-3 (pancreatic tumor type).
  • anti-HER3 Antibody A alone or in combination with cetuximab inhibited tumor growth of SCCHN A-253 xenograft tumors ( Figure 4).
  • Anti-HER3 Antibody A in combination with cetuximab caused significant reduction in tumor size, as shown in the waterfall plot ( Figure 5).

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Abstract

La présente invention concerne des anticorps qui se lient à HER3 humain et qui peuvent être utiles pour le traitement du cancer en monothérapie et en association avec des inhibiteurs de la voie EGFR, en particulier les cancers entraînés par la signalisation de l'EGFR ou qui utilisent HER3 comme mécanisme de résistance, notamment le cancer du poumon, le cancer de la tête et du cou, le cancer colorectal, le cancer du pancréas, le cancer rénal, le cancer gastrique, les cancers du sein ou le carcinome hépatocellulaire.
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