WO2009055074A2 - Erbb2 binding proteins and use thereof - Google Patents

Erbb2 binding proteins and use thereof Download PDF

Info

Publication number
WO2009055074A2
WO2009055074A2 PCT/US2008/012212 US2008012212W WO2009055074A2 WO 2009055074 A2 WO2009055074 A2 WO 2009055074A2 US 2008012212 W US2008012212 W US 2008012212W WO 2009055074 A2 WO2009055074 A2 WO 2009055074A2
Authority
WO
WIPO (PCT)
Prior art keywords
s1r3a1
bmv
erbb2
s1r2a
binding
Prior art date
Application number
PCT/US2008/012212
Other languages
French (fr)
Other versions
WO2009055074A3 (en
WO2009055074A8 (en
Inventor
Davinder Gill
Fionnuala Mcaleese
Maximillian Follettie
Laird Bloom
Peter A. Thompson
Peter R. Baum
Paul A. Algate
Original Assignee
Wyeth
Trubion Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2008/006905 external-priority patent/WO2008150485A2/en
Application filed by Wyeth, Trubion Pharmaceuticals, Inc. filed Critical Wyeth
Publication of WO2009055074A2 publication Critical patent/WO2009055074A2/en
Publication of WO2009055074A8 publication Critical patent/WO2009055074A8/en
Publication of WO2009055074A3 publication Critical patent/WO2009055074A3/en

Links

Classifications

    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • 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
    • C07K2317/74—Inducing cell proliferation
    • 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

  • This invention relates to binding proteins that bind erythroblastic leukemia viral oncogene homolog 2 (ErbB2), in particular, human ErbB2 (also known as HER2), and their use in regulating ErbB2-associated activities.
  • the binding proteins disclosed herein are useful in diagnosing, preventing, and/or treating ErbB2 associated disorders, e.g., hyperproliferative disorders, including cancer, and autoimmune disorders, including arthritis.
  • the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival.
  • the receptor family includes four distinct members including epidermal growth factor receptor (EGFR or ErbB1 ), HER2 (ErbB2 or p185 neu ), .
  • the ErbB receptors possess an extracellular domain (with four subdomains, I — IV), a single hydrophobic transmembrane domain, and (except for HER3) a highly conserved tyrosine kinase domain. Crystal structures of EGFR reveal a receptor that adopts one of two conformations.
  • EGFR In the "closed” conformation, EGFR is not bound by ligand and the extracellular subdomains Il and IV remain tightly apposed, preventing inter-receptor interactions. Ligand binding prompts the receptor to adopt an "open” conformation, in which the EGFR receptor is poised to make inter-receptor interactions.
  • the ErbB receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of ErbB ligands.
  • EGFR is bound by at least six different ligands; epidermal growth factor (EGF) 1 transforming growth factor alpha (TGF- ⁇ ), amphiregulin, heparin binding epidermal growth factor (HB-EGF), betacellulin and epiregulin.
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor alpha
  • HB-EGF heparin binding epidermal growth factor
  • betacellulin betacellulin
  • a family of heregulin proteins resulting from alternative splicing of a single gene are ligands for ErbB3 and ErbB4.
  • the heregulin family includes alpha, beta and gamma heregulins, neu differentiation factors (NDFs), glial growth factors (GGFs); acetylcholine receptor inducing activity (ARIA); and sensory and motor neuron derived factor (SMDF).
  • NDF neu differentiation factors
  • GGFs glial growth factors
  • ARIA acetylcholine receptor induc
  • HER2 was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats.
  • the activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein.
  • Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis.
  • Overexpression of ErbB2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
  • HER2 has been suggested to be a ligand orphan receptor.
  • the intracellular signaling pathway of HER2 is thought to involve ras-MAPK and PI3K pathways, as well as MAPK-independent S6 kinase and phospholipase C-gamma signaling pathways.
  • HER2 signaling also effects proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and an antiangiogenic factor, thrombospondin-1 (TSP-1).
  • VEGF vascular endothelial growth factor
  • IL-8 interleukin-8
  • TSP-1 thrombospondin-1
  • the full-length ErbB2 receptor undergoes proteolytic cleavage releasing its extracellular domain (ECD), which can be detected in cell culture medium and in patient's sera.
  • ECD extracellular domain
  • the truncated ErbB2 receptor (p95ErbB2) that remains after proteolytic cleavage exhibits increased autokinase activity and transforming efficiency compared with the full- length receptor, implicating the ErbB2 ECD as a negative regulator of ErbB2 kinase and oncogenic activity.
  • a recombinant humanized version of the murine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821 ,337) is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)).
  • HERCEPTIN® reportedly targets the C-terminal region of domain IV of ErbB2.
  • HERCEPTIN® clinical activity is predominately dependent on antibody dependent cell mediated cytotoxicity (ADCC). Studies have suggested that HERCEPTIN® acts by triggering G1 cell cycle arrest.
  • ErbB-directed therapeutics do not meet the current medical needs. ErbB-directed therapeutics have had only modest anti-tumor efficacy and are not as potent as anticipated from preclinical models. In most patients who initially respond to HERCEPTIN®, disease progression is noted within 1 year. In the metastatic setting, a median duration of roughly nine months was reported, at which point it appears that patients frequently become refractory to therapy. Studies have suggested that more complete blockade of the ErbB receptor family would be beneficial. As there are multiple functional domains of HER2, agents targeted to each of the domains could be a potentially valuable therapeutic. Additionally, there are harmful side effects of HERCEPTIN® treatment.
  • LVEF left ventricular ejection fraction
  • the invention relates to novel ErbB2 binding proteins that bind the extracellular domain (ECD) of ErbB2, in particular, human ErbB2.
  • the novel binding protein can be antibody, an antigen-binding fragment of an antibody or a small modular immunopharmaceutical (SMIP).
  • the binding proteins bind the ECD in the L1 , CR1 , L2 or CR2 domain, in some cases in the membrane proximal region of the CR2 domain, such as a membrane proximal region comprising the amino acid sequence shown in the first 12 residues of SEQ ID NO: 671 (i.e., without the EKK).
  • a HER2 binding protein of the invention is an ErbB2 agonist, increases tyrosine phosphorylation of ErbB2 and/or of AKT, MAP kinase (MAPK), MEK kinase, ERK 1/2, preferentially binds ErbB2 ECD homodimer over monomer or shed ECD, binds HER2 on cells and in some cases internalizes, decreases shedding of ErbB2 ectodomain shedding compared to shedding from cells of the same type without a bound HER2 binding protein of the invention, reduces the amount of cell surface HER2, reduces ErbB2 mediated proliferation of cancer cells, increases apoptosis in cancer cells, increases the number of cells in S phase after treatment with the binding protein, reduces tumor growth in vivo, enhances the effectiveness of some other anti-proliferative or cytotoxic agents or any combination of these properties.
  • MAPK MAP kinase
  • MEK kinase MEK kina
  • the invention further relates to nucleic acids encoding the binding proteins or their components, vectors and host cells comprising the nucleic acids and methods of producing the binding proteins by expressing them in the host cells.
  • kits and compositions comprising one or more binding proteins of the invention and in some embodiments, further comprising an additional component that is a therapeutic or diagnostic agent, particularly a chemotherapeutic agent.
  • the invention also provides methods for producing and identifying binding proteins of the invention and methods for using them, including for treating cancer or other ErbB2 mediated disorders in a subject in need thereof, for reducing proliferation of and/or increasing apoptosis in ErbB2 expressing cells, including cancer cells, for reducing tumor growth and for diagnostic uses, including detecting and/or quantifying the presence of ErbB2 or cells expressing it.
  • Figure 1 Schematic representation of the selection strategy used in the generation of human anti-Her2 scFv binding domains.
  • Figure 2 (A-M). Alignments of the heavy chain amino acid sequences of human anti-Her2 scFvs with the germline human V H gene sequence. CDRs are in bold type.
  • Figure 3 Alignments of the light chain amino acid sequences of human anti-Her2 scFvs with the germline human V ⁇ or V ⁇ sequence. CDRs are in bold type.
  • FIG. 1 Schematic diagram of the protein constructs used for selection and screening of scFvs and SMIPs that bind to the extracellular domain of Her2.
  • B scFvs and SMIPs are binned into 4 distinct groups according to their binding phenotype as determined using the reagents in Fig 4A. ( * Herceptin contact sites)
  • FIG. 5 ELISA data for scFv binding to Her2. Binding data for phage- expressed scFv binding to Her2-expressing cells is shown on the left side of the table and data for soluble scFv binding to purified Her2 proteins is shown on the right. ELISA data is scored using a range that correlates with binding signal as indicated by -, + etc.
  • FIG. 1 Binding of HER2 SMIPs (HER067 and HER030), HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) to (A) HER2 dimer; (B) HER2 monomer; and (C) HER2 shed ectodomain found in SKBR3 supernatant.
  • FIG. 7 ELISA and BIACORE® data for HERCEPTIN® (trastuzumab) and SMIPs binding to Her2.
  • Graphs represent binding of HERCEPTIN® (trastuzumab), HerO33 or Her030 binding to various Her2 proteins determined by standard ELISA methods.
  • the table represents Kd values for HERCEPTIN® (trastuzumab), HerO33, Her030 and HerO18 (Herceptin SMIP) binding to various Her2 proteins as detected by BIACORE®.
  • Figure 8 provides a summary of various specific SMIPs, HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) binding to various HER2 molecules (different sizes and different species, including human, murine, and macaque) as well as binding to several different cancer cell lines.
  • Figures 9A-9H show cell surface binding of HER2 SMIPs (HER067 and
  • HER094), HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) to cell lines
  • A Ramos (Her27CD20 + control);
  • B BT474;
  • C 22rv1 ;
  • D MDA-MB-175;
  • E MDA-MB-361 (ATCC);
  • F MDA-MB-453;
  • G MDA-MB-361 (JL); and
  • H SKBR3.
  • Figure 10 provides a summary of the anti-proliferative activity of HER033 SMIP and HERCEPTIN ® (trastuzumab) on several different cancer cell lines.
  • FIG. 11 Proliferation of MDA-MB-361 cells following treatment with HER030 or HER033.
  • MDA-MB-361 (ATCC) breast cancer cells were plated in 96-well format and treated with 0-10 ug/ml anti-Her2 or control reagents for 72 hr. Cells were washed, fixed, and stained with DAPI. Stained nuclei were counted using Cellomics High Content assay measuring fluorescence at 36OnM.
  • Figure 12 provides a summary of the anti-proliferative activity of various specific SMIPs, HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) on several different cancer cell lines.
  • FIG. 13 Western blot analysis of effect of HerO33 on Her2 receptor phosphorylation (Y1248) following 24hr treatment of MDA-MB-361 breast cancer cells.
  • Cells were treated in vitro with HerO33, HERCEPTIN® (trastuzumab), or a small molecule Her2 kinase inhibitor for 24hrs either alone or in the presence of heregulin (HRG1 10ng/ml) activation of Her3.
  • Protein lysates (50ug/well) were size fractionated by SDS-PAGE, transferred to nitrocellulose and probed with anti-phospho-Her2(Y1248) antibody. Inhibition of the Her2 receptor kinase blocked the endogenous Her2 autophosphorylation at tyrosine 1248 relative to control.
  • HerO33 increases downstream phosphoprotein signal transduction in MDA-MB-361 and BT474 breast cancer cells. Cells were plated in 96-well format and treated with anti-Her2 reagents or Heregulin for 10 minutes. Cells were stained with either rabbit anti-pAKT, anti-pERK, anti-pS6K, or anti-p38MAPK antibodies and ALEXA594 labeled secondary antibody and cellular fluorescence quantified by high content (Cellomics) analysis.
  • FIG. 15 Kinetic analysis of HerO33 stimulated downstream effector phosphorylation in MDA-MB-361 breast cancer cells.
  • Cells were grown in 96-well format and treated with either anti-Her2 reagents or Her3 ligand Heregulin for 10min to 24hr as indicated.
  • Cells were stained with either rabbit anti-pAKT, anti-pERK, anti-pS6K, or anti- p38MAPK antibodies and ALEXA594 labeled secondary antibody and cellular fluorescence quantified by high content (Cellomics) analysis.
  • HerO33 treatment induces sustained activation of AKT, ERK and p38MAP kinase phosphorylation in this cell line similar in magnitude to levels following stimulation with 10ng/ml Heregulin.
  • Figures 16A and 16B show level of phosphorylation of ErbB2, and ERK1/2 in MDA-MB-361 cells when treated with HER2 SMIP HER067, HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018).
  • Figure 17 shows the effect on cell cycle of HER033 SMIP, HERCEPTIN® (trastuzumab), and heregulin on the SKBR3 and BT474 cell lines.
  • Figure 18 shows the effect on cell cycle of HER033 SMIP, HERCEPTIN® (trastuzumab), and heregulin on the MDA-MB-453 and MDA-MB-361 cell lines.
  • FIG. 19 MDA-MB-361 xenograft progression in irradiated nu/nu mice.
  • Female nu/nu mice were exposed to 400 rads of total body irradiation. After three days, they were injected subcutaneously in the dorsal right flank with 1x10 7 MDA-MB-361 cells in Matrigel.
  • Figure 20 MDA-MB-361 xenograft progression in Balb/c nude mice. Male
  • mice Balb/c nude mice were injected subcutaneously in the dorsal right flank with 1x10 7 MDA-MB- 361 cells in Matrigel.
  • Figures 21 and 22 show the in vivo efficacy of HER2 SMIP
  • HER033/HER067 when used to treat SCID-Beige having a tumor xenograft of MDA-MB-361 cells and the in vitro anti-proliferative activity on MDA-MB-361 cells.
  • the bottom panel of Figure 21 shows a titration of anti-proliferative activity of HER2 SMIPs (HER067 and HER094) and trastuzumab
  • FIG. 22 shows the tumor volume of individual mice in each treatment group.
  • Figure 23 Alignments of the heavy chain amino acid sequences of human anti-ERBB2 antibodies with the germline human V H gene sequence. CDRs are in bold type.
  • Figure 24 Alignments of the light chain amino acid sequences of human anti-ERBB2 antibodies with the germline human V x or V ⁇ sequence. CDRs are in bold type.
  • Figures 25A and 25B are schematic representation of the
  • FIG. 25B shows the predicted structure of the "stumpy peptide” used for selection.
  • the EKK sequence at C terminus maintains the helical structure predicted from the NMR (Goetz et al.,
  • Figure 26 Alignments of the heavy chain and light chain amino acid sequences of human anti-ERBB2 antibodies with the germline human V H gene sequence.
  • FIG. 27 shows various HER2 soluble protein constructs used to investigate binding of molecules of the invention.
  • Figure 28 provides a summary of various specific SMIPs 1 HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) binding to various HER2 molecules (different sizes and different species, including human, murine, and macaque) as well as binding to Her2 monomers and shed extracellular domain.
  • Figure 29 is a graphical representation of different SMIPs binding to various Her2 molecules.
  • Figure 30 graphically depicts the binding of anti-HER2 "stumpy" binders (HER085, HER156 and HER 169) to soluble HER2 constructs.
  • Figure 31 summarizes the cell surface binding of various HER2 SMIPs to different cell lines.
  • Figure 32 is a bar graph showing cell staining of JIMT-1 cells with severalanti-HER2 SMIPS including "stumpy" binders.
  • Figure 33 graphically depicts staining of various cell lines with HER146,
  • Figure 34 summarizes the cross-reactivity of various HER2 SMIPs to Macaca Her2 and Murine Her2.
  • Figure 35 shows BIACORE® data for HERCEPTIN® (trastuzumab) and SMIPs binding to soluble Her2 proteins.
  • Figure 36 shows a titration of anti-proliferative activity of HER2 SMIPs (Her147, HeM 02, HeM 24, HerO67, HeM 46, HeM 16, HerO94, and HeM 33), trastuzumab SMIP (HER018) and Herceptin on MDAMB361 (ATCC) cells.
  • Figure 37 shows a titration of anti-proliferative activity of HER2 SMIPs (HeM 46, HerO67, HerO94, and HeM 16), trastuzumab SMIP (HER018) and Herceptin on MDAMB361 (JL) cells.
  • Figure 38 is a graph showing decreased proliferation of :MDA_MB-361 cells by anti-HER2 SMIPS HER146 and HER116.
  • Figure 39 is a table summarizing the anti-proliferative activity of various specific SMIPs, HERCEPTIN® (trastuzumab), and trastuzumab SMIP (HER018) on several different cancer cell lines.
  • Figure 40 is a graph showing the effect of MEK kinase inhibitor (CL-1040) on anti-HER2 SMIP anti-proliferative activity in MDA-MB-361 ATCC breast cancer cells.
  • Figure 41 is a graph showing the effect of ERK1/2 kinase inhibitor (FR180204) on anti-HER2 SMIP anti-proliferative activity in MDA-MB-361 ATCC breast cancer cells.
  • Figure 42 is a graph showing the effect of ERK1 or ERK2 knockdown by RNA interference on anti-HER2 SMIP anti-proliferative activity in MDA-MB-361 ATCC breast cancer cells.
  • Figure 43 is an image of a Western blot showing the presence of phosphorylated HER2 at 24 hrs and 48 hrs after treatment of MDA-MB-361 ATCC breast cancer cells with HER033 SMIP or HER146 SMIP.
  • Figures 44A and 44B show the effect on cell cycle of various SMIPs on the
  • Figures 45A-E show the effect on cell cycle of various SMIPs (A) MDA-MB- 453 (24 hours), (B) MDA-MB-361 (JL) (24 hours), (C) MDA-MB-361 (JL) (48 hours), (D) MDA-MB-361 (ATCC) (24 hours), (E), and MDA-MB-361 (ATCC) (48 hours). Samples in bold are statistically higher than the controls. Samples followed by " ** " are statistically lower than the controls (student T test with an error rate of 0.05).
  • Figure 46 is a graph of the mean tumor volume over time after treatement in vivo with anti-HER2 SMIPs HER146 and HER116 in SCID-Beige mice having an MDA- MB-361 (JL) cells tumor xenograft.
  • HERCEPTIN® tacuzumab
  • IgG vehicle
  • Figure 47 presents results in SCID-Beige mice having a tumor xenograft of MDA-MB-361 (JL) cells following treatment with HER146 SMIP and HER116 SMIP.
  • the left panel shows the survival of mice treated with HER146 SMIP, HER116 SMIP, HERCEPTIN® (trastuzumab), or vehicle (IgG) over a timecourse of 60 days.
  • the right panel shows tumor free progression of mice treated with HER146 SMIP, HER116 SMIP, HERCEPTIN® (trastuzumab), or vehicle (IgG) over a timecourse of 60 days.
  • the chart at the bottom demonstrates the mean survival time of mice used in the study.
  • Figures 48A-D are a set of graphs of MDA-MB-361 xenograft tumor size in
  • FIGS. 49A-D are a set of graphs of MDA-MB-361 xenograft tumor size in irradiated nu/nu mice after treatment with anti-HER2 SMIP HER146.
  • HERCEPTIN® (trastuzumab) and vehicle (IgG) are positive and negative controls, respectively.
  • A summary of data from 10 mice in each treatment group;
  • B data for individual mice in vehicle (negative control) group;
  • C data for individual mice in HER146 treatment group;
  • D data for individual mice in HERCEPTIN® (positive control) group.
  • Figure 50 presents data from two independent experiments investigating the effect of anti-HER2 SMIPS of the invention on the shedding of HER2 ectodomain and on HER2 cell surface expression.
  • (A) and (B) present the relative effect of various anti-HER2 SMIPS on ECD shedding as detected by ELISA.
  • Panels (C) and (D) presents the relative effect of various anti-HER2 SMIPS on HER2 expression.
  • Figure 51 presents data from the anti-HER2 SMIP cross-blocking experiments.
  • Figure 52 is a chart summarizing the cross-blocking results.
  • Figures 53 provide photographs depicting the internalization of anti-HER2 SMIP (panels A and B) and cell surface HER2 (panel C).
  • Figure 54 is a graph depicting Fc dependent cellular cytoxicity (FcDCC) of various anti-HER2 SMIPS in MDA-MB-361 -JL and SKBR3 cells.
  • Figure 55 is a graph depicting complement-dependent cytotoxicity (CDC)
  • Figure 56 presents data from ELISA testing of SMIP binding to Her2-SIIS after storage of the SMIP in plasma at various temperatures and durations.
  • A HerO67
  • B HeM 46.
  • Figures 57 depict different possible ratios of SM IP/receptor complexes with their predicted mass.
  • Figure 58 shows the masses of SMIP/receptor complexes observed following SEC-RI-MALLS analysis.
  • Figures 59A-D provide a series of dose response curves of different cells pre-treated with 5-fold dilution series of HER146 and then treated with corresponding 5-fold dilution series of different chemotherapeutic agents, or combinations thereof, and charts of the dilution series times of incubation used.
  • A MDA-MB-453 cells with HER146 and Cisplatin or Taxol
  • B MDA-MB-453 cells with HER146 and Doxorubicin
  • C MDA-MB-361- JL cells with Cisplatin or Taxol
  • D MDA-MB-361 -JLcells with HER146 and Doxorubicin or Gemcitabine.
  • Figure 60 is an immunoblot with short (left) or long (right) exposures showing Her2 immunoprecipitated from Ramos or SKBR3 cell lysates by Herceptin, 3B5, HER156, or HER169.
  • Figure 61 is two immunoblots in color and a black-and-white exposure of the color blot on the right, showing Her2 immunoprecipitated from Ramos, JIMT-1 , or MDA- MB-361 ATCC cell lysates by human IgG 1 3B5, HER116, HER156, or HER169.
  • the binding protein is an antibody or an antigen binding fragment of such antibody that specifically binds the ECD.
  • the binding protein is a small modular immunopharmaceutical (SMIP).
  • an antibody refers to an intact four-chain molecule having 2 heavy chains and 2 light chains, each heavy chain and light chain having a variable domain and a constant domain, or an antigen-binding fragment thereof, and encompasses any antigen- binding domain.
  • an antibody of the invention may be polyclonal, monoclonal, monospecific, polyspecific, bi-specific, humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted (including CDR grafted), or an in vitro generated antibody.
  • antigen-binding fragment of an antibody that specifically binds the ECD of ErbB2 refers to a portion or portions of the antibody that specifically binds to the ECD.
  • An antigen-binding fragment may comprise all or a portion of an antibody light chain variable region (V L ) and/or all or a portion of an antibody heavy chain variable region (V H ) so long as the portion or portions are antigen-binding. However, it does not have to comprise both. Fd fragments, for example, have two V H regions and often retain some antigen-binding function of the intact antigen-binding domain.
  • antigen-binding fragments of an antibody examples include (1 ) a Fab fragment, a monovalent fragment having the V L , V H , C L and C H 1 domains; (2) a F(ab') 2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) a Fd fragment having the two V H and C H 1 domains; (4) a Fv fragment having the V L and V H domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), that has a V H domain; (6) an isolated complementarity determining region (CDR) 1 and (7) a single chain Fv (scFv).
  • a Fab fragment a monovalent fragment having the V L , V H , C L and C H 1 domains
  • F(ab') 2 fragment a bivalent fragment having two Fab fragments linked by a disulfide bridge at the
  • V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883).
  • scFv single chain Fv
  • the term "effective amount” refers to a dosage or amount that is sufficient to alter ErbB2 activity, to ameliorate clinical symptoms or achieve a desired biological outcome, e.g., decreased cell growth or proliferation, decreased heterodimerization with another member of the EGF family decreased homodimerization, decrease tumor growth rate or tumor size, increased cell death etc.
  • human antibody includes antibodies having variable and constant region sequences corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (See Kabat, et al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The amino acid sequences of a human antibody, when aligned with germline immunoglobulin sequences, most closely align with human immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Such non-germline residues may occur in a framework region, a CDR, for example in the CDR3, or in the constant region.
  • a human antibody can have one or more residues, such as any number from 1-15, including all of the integers between 1 and 15, or more, replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.
  • CDRs are as defined by Kabat or in Chothia C, Lesk AM, Canonical structures for the hypervariable regions of immunoglobulins, J MoI Biol.
  • the phrase "inhibit” or “antagonize” an ErbB2/HER2 activity refers to a reduction, inhibition, or otherwise diminution of at least one activity of ErbB2 due to binding an anti-ErbB2 antibody or antigen binding portion, wherein the reduction is relative to the activity of ErbB2 in the absence of the same antibody or antigen-binding portion.
  • the activity can be measured using any technique known in the art, including, for example, as described in the Examples.
  • Activation of the Her2 receptor tyrosine kinase can be measured by the degree of phosphorylation of key tyrosine residues in the intracellular domain.
  • Tyr1248 is a known site of autophosphorylation and thus is a direct measure of Her2 receptor kinase activity.
  • the degree of phosphorylation can be determined by Western blot analysis probing with anti-phopho-Her2 specific antibodies (eg. Tyr1248, Tyr1139, Tyr1112, Tyr877, Tyr1221/1222).
  • cells can be permeabilized and probed with fluorescently labeled phospho-Her2 antibodies and measured either by flow cytometry or high content (Cellomics) analysis.
  • the Her2 receptor can be immunoprecipitated, digested with trypsin protease and the degree of phosphorylation at specific sites within the individual Her2 peptides determined by standard Mass Spec techniques.
  • Inhibition or antagonism does not necessarily indicate a total elimination of the ErbB2 polypeptide biological activity.
  • the reduction in activity may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, including 100% reduction, i.e., elimination of the activity.
  • ErbB2 refers to erythroblastic leukemia viral oncogene homolog 2. In the case of human ErbB2, it also is known as c-erb-B2 or HER2/neu.
  • the ErbB2 may comprise: (1 ) an amino acid sequence of a naturally occurring mammalian ErbB2 polypeptide (full length or mature form) or a fragment thereof, or a fragment thereof; (2) an amino acid sequence substantially identical to, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to said amino acid sequence or a fragment thereof; (3) an amino acid sequence that is encoded by a naturally occurring mammalian ErbB2 nucleotide sequence or a fragment thereof, or (4) a nucleotide sequence that hybridizes to the foregoing nucleotide sequence under stringent conditions, e.g., highly stringent conditions.
  • HER2 or c-erb-B2 encodes a transmembrane receptor protein of 185 kDa, which is structurally related to the epidermal growth factor receptori .
  • HER2 protein overexpression is observed in 25%-30% of primary breast cancers and is associated with decreased overall survival and a lowered response to chemotherapy and hormonal therapy, which can continue throughout the course of the disease and drives aggressive tumor growth.
  • ErbB2 activity refers to at least one cellular process initiated or interrupted as a result of ErbB2 binding to a receptor complex comprising ErbB2 and an ErbB receptor family member including ErbB1 (EGFR), ErbB2, ErbB3, ErbB4 or comprising an ErbB ligand such as but not limited to EGF, TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 on the cell.
  • ErbB2 activity can be determined using any suitable assay methods, for example, protein overexpression can be determined using immunohistochemistry (IHC) and may also be inferred when HER2 gene amplification is identified using fluorescence in situ hybridization (FISH).
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization
  • in vitro generated antibody refers to an antibody where all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection (e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen). This term excludes sequences generated by genomic rearrangement in an immune cell.
  • isolated refers to a molecule that is substantially free of its natural environment.
  • an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived.
  • the term also refers to preparations where the isolated protein is sufficiently pure for pharmaceutical compositions; or at least 70-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
  • percent identical refers to the similarity between at least two different sequences. This percent identity can be determined by standard alignment algorithms, for example, the Basic Local Alignment Tool (BLAST) described by Altshul et al. ((1990) J. MoI. Biol., 215: 403-410); the algorithm of Needleman et al. ((1970) J. MoI. Biol., 48: 444-453); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4: 11-17). A set of parameters may be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • BLAST Basic Local Alignment Tool
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) that has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity is usually calculated by comparing sequences of similar length.
  • binding refers to forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the association constant K A is higher than 10 6 M "1 .
  • the appropriate binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques. An antibody is said to specifically bind an antigen when the K 0 is ⁇ 1 mM, preferably ⁇ 10O nM.
  • stringent describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • One example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2X SSC, 0.1% SDS at 50 0 C.
  • SSC sodium chloride/sodium citrate
  • a second example of stringent hybridization conditions is hybridization in 6X SSC at about 45°C, followed by at least one wash in 0.2X SSC, 0.1% SDS at 55°C.
  • stringent hybridization conditions hybridization in 6X SSC at about 45°C, followed by at least one wash in 0.2X SSC, 0.1 % SDS at 6O 0 C.
  • a further example of stringent hybridization conditions is hybridization in 6X SSC at about 45 0 C, followed by at least one wash in 0.2X SSC, 0.1% SDS at 65°C.
  • High stringent conditions include hybridization in 0.5M sodium phosphate, 7% SDS at 65°C, followed by at least one wash at 0.2X SSC 1 1 % SDS at 65°C.
  • substantially homologous means that the relevant amino acid or nucleotide sequence (e.g., CDR(s), V H , or V L domain) will be identical to or have insubstantial differences (through conserved amino acid substitutions) in comparison to the sequences that are set out. Insubstantial differences include minor amino acid changes, such as 1 or 2 substitutions in a 5 amino acid sequence of a specified region.
  • the second antibody has the same specificity and has at least 50% of the affinity of the first antibody.
  • sequences substantially identical or homologous e.g., at least about 85% sequence identity
  • sequence identity can be about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher.
  • substantial identity or homology exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • therapeutic agent is a substance that treats or assists in treating a medical disorder.
  • Therapeutic agents may include, but are not limited to, anti-proliferative agents, anti-cancer agents including chemotherapeutics, anti-virals, anti-infectives, immune modulators, and the like that modulate immune cells or immune responses in a manner that complements the ErbB2 activity of an anti-ErbB2 binding protein of the invention.
  • Non- limiting examples and uses of therapeutic agents are described herein.
  • a "therapeutically effective amount" of an anti-ErbB2 binding protein refers to an amount of an binding protein that is effective, upon single or multiple dose administration to a subject (such as a human patient) at treating, preventing, curing, delaying, reducing the severity of, and/or ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.
  • treatment refers to a therapeutic or preventative measure.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay, reduce the severity of, and/or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • the invention provides novel ErbB2/HER2, particularly human ErbB2/HER2, ErbB2/HER2 binding proteins that bind in the extra-cellular domain
  • the binding proteins of the invention bind in the LR1 , CR1 , LR2 or CR2 domain of the ECD, including a membrane proximal region of CR2 comprising the amino acid sequence in the first twelve residues of SEQ ID NO: 671 (i.e., without the EKK).
  • the binding proteins of the invention preferentially bind ErbB2 nomodimers over monomers or shed ECD.
  • the binding proteins of the invention bind ECD homodimers substantially more than monomers. In some cases, the binding protein has no appreciable or significant binding to ECD monomers or to shed ECD.
  • the novel binding proteins are ErbB2 agonists and increase tyrosine phosphorylation of ErbB2 and at the same time, have anti-proliferative activity and pro-apoptotic activity.
  • the binding protein increases kinase activity in a HER-2 expressing cell, including but not limited to increasing kinase activity of MEK, MAPK, ERK1 , ERK2 or a combination thereof.
  • the anti-ErbB2/HER2 binding proteins of the invention can be obtained by any of numerous methods known to those skilled in the art.
  • antibodies can be produced using recombinant DNA methods (U.S. Patent 4,816,567).
  • Monoclonal antibodies may be produced by generation of hybridomas (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499) in accordance with known methods.
  • Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORETM) analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen.
  • ELISA enzyme-linked immunosorbent assay
  • BIACORETM surface plasmon resonance
  • any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.
  • One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315- 1317; Clackson et al. (1991 ) Nature, 352: 624-628; Marks et al. (1991) J. MoI.
  • the specified antigen can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat.
  • the non-human animal includes at least a part of a human immunoglobulin gene.
  • antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSETM, Green et al.
  • a complete 4-chain immunoglobulin comprises active portions, e.g., a portion of the V H or V L domain or a CDR that binds to the antigen, i.e., an antigen-binding fragment, or, e.g., the portion of the C H subunit that binds to and/or activates, e.g., an Fc receptor and/or complement.
  • CDRs typically refer to regions that are hypervariable in sequence and/or form structurally defined loops, for example, Kabat CDRs are based on sequence variability, as described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds.
  • a monoclonal antibody is obtained from the non- human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art.
  • modified e.g., humanized, deimmunized, chimeric
  • a variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.
  • Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539).
  • All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.
  • Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; by Oi et al.
  • BioTechniques ⁇ WA and by US 5,585,089; US 5,693,761 ; US 5,693,762; US 5,859,205; and US 6,407,213.
  • Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain.
  • nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources.
  • the recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.
  • a humanized antibody is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations.
  • altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymoi, 92: 3-16, 1982), and may be made according to the teachings of PCT Publication WO92/06193 or EP 0239400).
  • An antibody or fragment thereof may also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317).
  • peptide threading For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class Il binding peptides can be searched for motifs present in the V H and V L sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class Il DR allotypes, and thus constitute potential T cell epitopes.
  • Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used.
  • an antibody can contain an altered immunoglobulin constant or Fc region.
  • an antibody produced in accordance with the teachings herein may bind more strongly or with more specificity to effector molecules such as complement and/or Fc receptors, which can control several immune functions of the antibody such as effector cell activity, lysis, complement-mediated activity, antibody clearance, and antibody half-life.
  • Typical Fc receptors that bind to an Fc region of an antibody include, but are not limited to, receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RI 11 and FcRn subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc receptors are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92, 1991 ; Capel et al., lmmunomethods 4:25-34,1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41 , 1995).
  • an anti-ErbB2 antibody of the invention may be a
  • V HH molecules are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains, such as those derived from Camelidae as described in WO9404678, incorporated herein by reference.
  • Such a V HH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco and is sometomes called a camelid or camelized variable domain. See e.g., Muyldermans., J. Biotechnology (2001 ) 74(4):277-302, incorporated herein by reference.
  • V HH molecules are about 10 times smaller than IgG molecules. They are single polypeptides in which the CDR3 is longer than a conventional antibody, the VH:VL interface residues are different, and extra cysteines are generally present. These molecules tend to be very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of V HH S produces high yield, properly folded functional V HH S.
  • an anti-ErbB2 antibodies or binding fragments of the invention may include single domain antibodies such as immunoglobulin new antigen receptors (IgNARs), which are a unique group of antibody isotypes found in the serum of sharks
  • IgNARs immunoglobulin new antigen receptors
  • Antibodies also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D 1 E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgGI , lgG2, lgG3, lgG4, IgAI , and lgA2.
  • subclasses e.g., IgGI , lgG2, lgG3, lgG4, IgAI , and lgA2.
  • Each light chain includes an N terminal variable (V) domain (V L ) and a constant (C) domain (Q_).
  • Each heavy chain includes an N terminal V domain (V H ), three or four C domains (C H s), and a hinge region collectively referred to as the constant region of the heavy chain.
  • the C H domain most proximal to V H is designated as C H 1.
  • the V H and V L domains consist of four regions of relatively conserved sequences called framework regions (FR1 , FR2, FR3, and FR4), that form a scaffold for three regions of hypervariable sequences also referred to as complementarity determining regions CDRs.
  • CDRs are referred to as CDR1 , CDR2, and CDR3.
  • CDR constituents on the heavy chain may be referred to as HCDR1 , HCDR2, and HCDR3, while CDR constituents on the light chain are referred to as LCDR1 , LCDR2, and LCDR3.
  • CDR3 is typically the greatest source of molecular diversity within the antibody-binding site.
  • the anti-ErbB2 binding proteins of the invention include complete 4-chain antibodies and antigen-binding fragments of complete antibodies.
  • An antigen-binding fragment also referred to as an antigen-binding portion
  • the Fab fragment (Fragment antigen-binding) consists of V H -CH1 and V L -C L domains covalently linked by a disulfide bond between the constant regions.
  • the F v fragment is smaller and consists of V H and V L domains non-covalently linked.
  • a single chain F v fragment (scF v ) can be constructed.
  • the scF v contains a flexible polypeptide that links (1) the C-terminus of V H to the N-terminus of V L , or (2) the C-terminus of V L to the N-terminus of V H . Repeating units of (Gly 4 Ser)_often 3 or 4 repeats may be used as a linker, but other linkers are known in the art.
  • a "bispecific” or “bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab 1 fragments. See, e.g., Songsivilai & Lachmann, CHn. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).
  • the bispecific antibody comprises a first binding domain polypeptide, such as a Fab' fragment, linked via an immunoglobulin constant region to a second binding domain polypeptide.
  • an anti-ErbB2 binding protein of the invention is a Small Modular jmmunoPharmaceuticals (SMIPTM).
  • SMIPs and their uses and applications are disclosed in, e.g., U.S. Published Patent Application. Nos. 2003/0118592, 2003/0133939, 2004/0058445, 2005/0136049, 2005/0175614, 2005/0180970, 2005/0186216, 2005/0202012, 2005/0202023, 2005/0202028, 2005/0202534, and 2005/0238646, and related patent family members thereof, all of which are hereby incorporated by reference herein in their entireties.
  • a SMIPTM typically refers to a binding domain-immunoglobulin fusion protein that includes a binding domain polypeptide that is fused or otherwise connected to an immunoglobulin hinge or hinge-acting region polypeptide, which in turn is fused or otherwise connected to a region comprising one or more native or engineered constant regions from an immunoglobulin heavy chain, other than C H 1 , for example, the C H 2 and C H 3 regions of IgG and IgA, or the C H 3 and C H 4 regions of IgE (see e.g., U.S. 2005/0136049 by Ledbetter, J. et a/., which is incorporated by reference, for a more complete description).
  • the binding domain-immunoglobulin fusion protein can further include a region that includes a native or engineered immunoglobulin heavy chain C H 2 constant region polypeptide (or C H 3 in the case of a construct derived in whole or in part from IgE) that is fused or otherwise connected to the hinge region polypeptide and a native or engineered immunoglobulin heavy chain C H 3 constant region polypeptide (or C H 4 in the case of a construct derived in whole or in part from IgE) that is fused or otherwise connected to the C H 2 constant region polypeptide (or C H 3 in the case of a construct derived in whole or in part from IgE).
  • a native or engineered immunoglobulin heavy chain C H 2 constant region polypeptide or C H 3 in the case of a construct derived in whole or in part from IgE
  • C H 4 native or engineered immunoglobulin heavy chain C H 3 constant region polypeptide
  • binding domain-immunoglobulin fusion proteins are capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytotoxicity, complement fixation, and/or binding to a target, for example, a target antigen, such as human ErbB2.
  • the binding domain of a SMIP of the invention may contain a complete V H and a complete V L joined by linker antigen-binding portions of a V H and/or V L and may V2 or be linked in either orientation, i.e., V H -linker-V L or V L -linker-V H .
  • Any suitable linker can be used in a SMIP of the invention and will be known to those of skill in the art. Exemplary linkers may be found, for example in WO 2007/146968 Tables 5 and 10-12 of which are incorporated by reference in their entirety.
  • any immunoglobulin hinge sequence or hinge-acting sequence may be used in a SMIP of the invention.
  • the immunoglobulin heavy chain constant region polypeptides is from a human immunoglobulin heavy chain.
  • the immunoglobulin heavy chain constant region polypeptides are of an isotype selected from human IgG and human IgA.
  • the linker polypeptide comprises at least one polypeptide having as an amino acid sequence (GIy 4 , Ser) and in certain other embodiments the linker polypeptide comprises at least three repeats of said polypeptide.
  • the immunoglobulin hinge region polypeptide comprises a human IgA hinge region polypeptide.
  • An immunoglobulin hinge region polypeptide includes any hinge peptide or polypeptide that occurs naturally, as an artificial peptide or as the result of genetic engineering and that is situated in an immunoglobulin heavy chain polypeptide between the amino acid residues responsible for forming intrachain immunoglobulin-domain disulfide bonds in CH 1 and CH2 regions; hinge region polypeptides for use in the present invention may also include a mutated hinge region polypeptide.
  • an immunoglobulin hinge region polypeptide may be derived from, or may be a portion or fragment of (i.e., one or more amino acids in peptide linkage, typically 5-65 amino acids, preferably 10-50, more preferably 15-35, still more preferably 18-32, still more preferably 20- 30, still more preferably 21 , 22, 23, 24, 25, 26, 27, 28 or 29 amino acids) an immunoglobulin polypeptide chain region classically regarded as having hinge function, as described above.
  • a hinge region polypeptide for use in the instant invention need not be so restricted and may include amino acids situated (according to structural criteria for assigning a particular residue to a particular domain that may vary, as known in the art) in an adjoining immunoglobulin domain such as a CH1 domain or a CH2 domain, or in the case of certain artificially engineered immunoglobulin constructs, an immunoglobulin variable region domain.
  • Wild-type immunoglobulin hinge region polypeptides include any naturally occurring hinge region that is located between the constant region domains, CH 1 and CH2, of an immunoglobulin.
  • the wild-type immunoglobulin hinge region polypeptide is preferably a human immunoglobulin hinge region polypeptide, preferably comprising a hinge region from a human IgG immunoglobulin, and more preferably, a hinge region polypeptide from a human IgGI isotype.
  • immunoglobulin primary structure exhibits a high degree of sequence conservation in particular portions of immunoglobulin polypeptide chains, notably with regard to the occurrence of cysteine residues which, by virtue of their sulfyhydryl groups, offer the potential for disulfide bond formation with other available sulfydryl groups.
  • wild-type immunoglobulin hinge region polypeptides may be regarded as those that feature one or more highly conserved (e.g., prevalent in a population in a statistically significant manner) cysteine residues, and in certain preferred embodiments a mutated hinge region polypeptide may be selected that contains zero or one cysteine residue and that is derived from such a wild-type hinge region.
  • a mutated immunoglobulin hinge region polypeptide may comprise a hinge region that has its origin in an immunoglobulin of a species, of an immunoglobulin isotype or class, or of an immunoglobulin subclass that is different from that of the CH2 and CH3 domains.
  • the SMIP may comprise a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide comprising a wild-type human IgA hinge region polypeptide, or a mutated human IgA hinge region polypeptide that contains zero or only one cysteine residues, as described herein.
  • Such a hinge region polypeptide may be fused to an immunoglobulin heavy chain CH2 region polypeptide from a different Ig isotype or class, for example an IgG subclass, which in certain preferred embodiments will be the IgGI subclass.
  • an anti-ErbB2 antibody of the invention is a V HH molecule.
  • V HH molecules (or nanobodies), as known to the skilled artisan, are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains, such as those derived from Camelidae as described in WO9404678, incorporated herein by reference.
  • Such a V HH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco and is sometomes called a camelid or camelized variable domain. See e.g., Muyldermans., J.
  • V HH molecules are about 10 times smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of V HH S produces high yield, properly folded functional V HH S. In addition, antibodies generated in Camelids will recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids (see WO 9749805, that is incorporated herein by reference).
  • Amino acid (AA) sequences of illustrative heavy chain variable domains (V H ) and light chain variable domains (V L ) of the anti-ErbB2 antibodies of this invention are set forth in the attached Sequence Table.
  • Table 1 provides the Sequence Identifiers (SEQ ID Nos) of the V H and V L domains.
  • S1 R2A_CS_1 F7 S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1 R2C_CS_1 H12, S1 R2A_CS_1 D3, S1 R3B2_BMV_1 E1 , S1 R3C1_CS_1 D3, S1 R3B2_DP47_1 E8, S1 R3B2_BMV_1 G2, S1R3B2_BMV_1 H5, S1 R3C1_CS_1A6, S1 R3B2_DP47_1C9, S1 R3B2_DP47_1 E10, S1 R3C1_CS_1 B10, S1R3A1_BMV_1 F3, S1 R3B1_BMV_1G11 , S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1
  • S1 R2A_CS_1 F7 indicates clone 1 F7 from round 2A of the first selection from the CS library.
  • An anti-ErbB2 binding protein of this invention may optionally comprise antibody constant regions or parts thereof.
  • a V L domain may be attached at its C-terminal end to a light chain constant domain which can be a CK or a C ⁇ .
  • a V H domain or portion thereof may be attached to all or part of a heavy chain constant region, which can be a IgA, IgD, IgE, IgG, or IgM constant region or any isotype subclass including IgGI, lgG2, lgG3, lgG4, IgAI or lgA2.
  • binding proteins within the scope of this invention may include V H and V L domains, or a portion thereof, combined with constant regions or portions thereof known in the art.
  • the ErbB2 binding protein comprises a V H domain, a V L domain, or a combination thereof, comprising the V H or V L amino acid sequence, respectively, found in any one of S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1R2C_CS_1D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3, S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV
  • An anti-ErbB2 antibody of the invention may comprise one, two, three, four, five or all six complementarity determining regions (CDRs) from any one of the above-listed antibodies.
  • an anti-ErbB2 binding protein of the invention comprises the HCDR1, HCDR2 and HCDR3 (heavy chain CDR set), the LCDR1, LCDR2 and LCDR3 (light chain CDR set) or both the heavy chain CDR set and the light chain CDR set of one of the anti-ErbB2 antibodies exemplified herein.
  • an anti-ErbB2 binding protein of the invention comprises an HCDR3 amino acid sequence found in any one of S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3, S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6,
  • S1R3B1_DP47_3A2 S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1 R3A1_DP47_11 F6, S1 R3A1_DP47_11 D3, S1 R3A1_CS_8A8, S1 R3A1_BMV_5
  • the V H and/or V L domains may be germlined, i.e., the framework regions (FR) of these domains are mutated using conventional molecular biology techniques to match the germline sequence.
  • the FR sequences remain diverged from the consensus germline sequences.
  • mutagenesis is used to make an antibody more similar to one or more germline sequences. This may be desirable when mutations are introduced into the framework region of an antibody through somatic mutagenesis or through error prone PCR.
  • Germline sequences for the V H and V L domains can be identified by performing amino acid and nucleic acid sequence alignments against the VBASE database (MRC Center for Protein Engineering, UK).
  • VBASE is a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences, including those in the current releases of the Genbank and EMBL data libraries.
  • the FR regions of the scFvs are mutated in conformity with the closest matches in the VBASE database and the CDR portions are kept intact.
  • an anti-ErbB2 binding of this invention specifically binds the same epitope as, competes with or cross-competes with an antibody selected from the group consisting of: S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3,
  • S1R3B2_DP47_1E8 S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1,
  • such competing or ErbB2-mediated cross- competing binding protein is an ErbB2 agonist and may further reduce proliferation of a cancer call, reduce the rate of growth of an ErbB2-expressing tumor and/or increases apoptosis in such cells and tumors.
  • such competing or cross- competing binding proteins bind ErbB2 ECD homo-dimers but do not bind ECD monomers or shed ECD.
  • Such antibodies can be identified in a competitive binding assay. One can determine whether an antibody binds to the same epitope or cross competes for binding with a binding protein of the invention antibody by using methods known in the art.
  • the association constant (K A ) of an ErbB2 binding protein of the invention is at least 10 6 M "1 .
  • the association constant of these antibodies for human ErbB2 is at least 10 9 M "1 .
  • the association constant of these antibodies for human ErbB2 is at least 10 10 M '1 , at least 10 11 M '1 , or at least 10 12 M '1 .
  • the binding affinity may be determined using techniques known in the art, such as ELISA, biosensor technology, such as biospecific interaction analysis, or other techniques including those described in this application.
  • epitope mapping see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996)
  • secondary and tertiary structure analyses can be carried out to identify specific 3D structures assumed by the presently disclosed antibodies and their complexes with antigens.
  • Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11 :7-13) and computer modeling of virtual representations of the present antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • the invention further provides anti-ErbB2 binding proteins that comprise altered V H and/or V L sequence(s) compared to the sequences in Table 1.
  • binding proteins may be produced by a skilled artisan using techniques well-known in the art. For example, amino acid substitutions, deletions, or additions can be introduced in FR and/or CDR regions.
  • FR changes are usually designed to improve the stability and immunogenicity of the antibody, while CDR changes are typically designed to increase antibody affinity for its antigen. The changes that increase affinity may be tested by altering CDR sequence and measuring antibody affinity for its target (see Antibody Engineering, 2nd ed., Oxford University Press, ed. Borrebaeck, 1995).
  • Antibodies whose CDR sequences differ insubstantially from those found in any one of specifically exemplified anti-ErbB2 antibodies are encompassed within the scope of this invention. Typically, this involves substitution of an amino acid with an amino acid having similar charge, hydrophobic, or stereochemical characteristics. More drastic substitutions in FR regions, in contrast to CDR regions, may also be made as long as they do not adversely affect (e.g., reduce affinity by more than 50% as compared to unsubstituted antibody) the binding properties of the binding protein. Substitutions may also be made to germline the binding protein or stabilize the antigen binding site.
  • Conservative modifications will produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are made.
  • substantial modifications in the functional and/or chemical characteristics of the molecules may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (1 ) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (2) the charge or hydrophobicity of the molecule at the target site, or (3) the size of the molecule.
  • a "conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • Desired amino acid substitutions can be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein.
  • Exemplary amino acid substitutions include, but are not limited to, those set forth in Table 2.
  • conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems.
  • the method for making a variant V H domain comprises adding, deleting, or substituting at least one amino acid in the disclosed V H domains, and testing the variant V H domain for ErbB2 binding or modulation of ErbB2 activity.
  • An analogous method for making a variant V L domain comprises adding, deleting, or substituting at least one amino acid in the disclosed V L domains, and testing the variant V L domain for ErbB2 binding or modulation of ErbB2 activity.
  • a further aspect of the invention provides a method for preparing antibodies or antigen-binding fragments that specifically bind ErbB2.
  • the method comprises:
  • At least one V L CDR or V H CDR of the invention is combined with a repertoire of nucleic acids encoding a V L or V H domain , respectively, that lacks at least one CDR or contains at least one CDR to be replaced.
  • the at least one V H or V L CDR may be a CDR1 , a CDR2, a CDR3, or a combination thereof, found in any of the specifically exemplified anti-ErbB2 antibodies.
  • variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and the at least one donor nucleic acid encodes a CDR3 amino acid sequence found in any one of SEQ ID Nos:1-95, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271, 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371 , 373, 375, 377, 3
  • variable domain includes a CDR1 to be replaced or lacks a CDR1 encoding region and the at least one donor nucleic acid encodes a CDR1 amino acid sequence found in any one of SEQ ID Nos: 1-95, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371 , 373, 375, 3
  • variable domain includes a CDR2 to be replaced or lacks a CDR2 encoding region and the at least one donor nucleic acid encodes a CDR2 amino acid sequence found in any one of SEQ ID Nos: 1-95, 251 , 253, 255, 257, 259, 261, 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321, 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371, 373, 375, 377, 379, 38
  • variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and further comprises a CDR1 to be replaced or lacks a CDR1 encoding region, where the at least one donor nucleic acid encodes a CDR3 a CDR1 amino acid sequence, respectively, found in any one of SEQ ID Nos: 1-95, 251 , 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,
  • variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and further comprises a CDR2 to be replaced or lacks a CDR2 encoding region, where the at least one donor nucleic acid encodes a CDR3 or CDR2 amino acid sequence, respectively, found in any one of SEQ ID Nos: 1-95, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 3
  • variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and further comprises a CDR1 and a CDR2 to be replaced or lacks a CDR1 and a CDR2 encoding region, where the at least one donor nucleic acid encodes CDR3, CDR1 or CDR2 amino acid sequence, respectively, found in any one of SEQ ID Nos: 1-95, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351, 353, 355, 3
  • the present invention further encompasses anti-ErbB2 antibodies comprising an HCDR3, an LCDR3 or both, three heavy chain CDRs, three light chain CDRs or all six CDRs, a V H or V L or an antigen-binding portion of such a V H or V L . or both, of a specifically provided molecule herein
  • a disclosed CDR sequence may be introduced into a repertoire of V H or V L domains lacking the respective CDR (Marks et al. (BioTechnology (1992) 10: 779-783).
  • a primer adjacent to the 5' end of the variable domain and a primer to the third FR can be used to generate a repertoire of variable domain sequences lacking CDR3.
  • This repertoire can be combined with a CDR3 of an antibody disclosed herein.
  • portions of a disclosed CDR sequence may be shuffled with portions of CDR sequences from other antibodies to provide a repertoire of antigen-binding fragments that bind ErbB2.
  • Either repertoire can be expressed in a host system such as phage display (described in WO 92/01047 and its corresponding U.S. Patent No. 5,969,108) so suitable antigen-binding fragments that bind to ErbB2 can be selected.
  • a host system such as phage display (described in WO 92/01047 and its corresponding U.S. Patent No. 5,969,108) so suitable antigen-binding fragments that bind to ErbB2 can be selected.
  • a further alternative uses random mutagenesis of a V H or V L sequence disclosed herein to generate variant V H or V L domains still capable of binding ErbB2.
  • a technique using error-prone PCR is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).
  • Another method uses direct mutagenesis of a V H or V L sequence disclosed herein. Such techniques are described by Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91 : 3809-3813) and Schier et al. (J. MoI. Biol. (1996) 263: 551-567).
  • variable domains that comprises at least one CDR region substantially as set out herein and, optionally, intervening framework regions from the V H or V L domains as set out herein.
  • Variable domains lacking a portion of the N-terminus of the FR1 and/or a portion of the Ci terminus of the FR4 are also encompassed by the invention. Additional residues at the N-terminal of the FR1 or C-terminal of the FR4 of the variable domain may not be the same residues found in naturally occurring antibodies. For example, construction of antibodies by recombinant DNA techniques often introduces N- or C-terminal residues from its use of linkers. Some linkers may be used to join variable domains to other variable domains (e.g., diabodies), constant domains, or proteinaceous labels.
  • embodiments specifically exemplified herein comprise a "matching" pair of V H and V L domains
  • alternative embodiments may comprise binding proteins containing only a single CDR from either V L or V H domain.
  • Either one of the V H domain or V L domain can be used to screen for complementary domains capable of forming a two-domain specific binding protein capable of, binding to ErbB2 ECD.
  • the screening may be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO 92/01047.
  • the anti-ErbB2 binding protein can be linked to a protein (e.g., albumin) by chemical cross-linking or recombinant methods.
  • the disclosed antibodies may also be linked to a variety of nonproteinaceous polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes) in manners set forth in U.S. Patent Nos.
  • binding proteins can be chemically modified by covalent conjugation to a polymer, for example, to increase their half-life in blood circulation.
  • Exemplary polymers and attachment methods are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.
  • Binding proteins of the invention can be modified to alter their glycosylation; that is, at least one carbohydrate moiety can be deleted or added to the binding protein, for example to modify antibody dependent (or Fc dependent) cellular cytotoxicity (ADCC/FcDCC), in particular to enhance ADCC/FcDCC.
  • ADCC/FcDCC antibody dependent cellular cytotoxicity
  • glycosylation sites can be accomplished by changing amino acid sequence to delete or create glycosylation consensus sites, that are well known in the art.
  • Another means of adding carbohydrate moieties is the chemical or enzymatic coupling of glycosides to amino acid residues of the antibody (see WO 87/05330 and ApNn et al.
  • Antibodies with altered function can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 A1 , US 5,624,821 and US 5,648,260). Similar types of alterations could be described that if applied to a murine or other species antibody would reduce or eliminate similar functions.
  • an Fc region of an antibody e.g., an IgG, such as a human IgG
  • FcR e.g., Fc gamma R1
  • C1q FcR
  • the affinity may be altered by replacing at least one specified residue with at least one residue having an appropriate functionality on its side chain, or by introducing a charged functional group, such as glutamate or aspartate, or perhaps an aromatic non-polar residue such as phenylalanine, tyrosine, tryptophan or alanine (see e.g., US 5,624,821).
  • residue 297 (asparagine)
  • alanine in the IgG constant region significantly inhibits recruitment of effector cells, while only slightly reducing (about three fold weaker) affinity for CIq (see e.g., US 5,624,821 ).
  • the numbering of the residues in the heavy chain is that of the EU index (see Kabat et al., 1991 supra). This alteration destroys the glycosylation site and it is believed that the presence of carbohydrate is required for Fc receptor binding. Any other substitution at this site that destroys the glycosylation site is believed to cause a similar decrease in lytic activity.
  • amino acid substitutions e.g., changing any one of residues 318 (GIu), 320 (Lys) and 322 (Lys), to Ala, are also known to abolish CIq binding to the Fc region of IgG antibodies (see e.g., US 5,624,821).
  • Modified binding proteins can be produced that have a reduced interaction with an Fc receptor.
  • Fc receptor For example, it has been shown that in human IgG 3 , which binds to the human Fc gamma R1 receptor, changing Leu 235 to GIu destroys its interaction with the receptor.
  • Mutations on adjacent or close sites in the hinge link region of an antibody e.g., replacing residues 234, 236 or 237 with Ala
  • the numbering of the residues in the heavy chain is based in the EU index (see Kabat et al., 1991 supra).
  • a binding protein of this invention may be tagged with a detectable or functional label.
  • labels include radiolabels (e.g., 131 I or 99 Tc), enzymatic labels (e.g., horseradish peroxidase or alkaline phosphatase), and other chemical moieties (e.g., biotin).
  • the invention features a human, monoclonal antibody that specifically binds the ECD, ErbB2, in particular, human ErbB2 and posseses onr or more of the following characteristics: (1) it is an in vitro generated antibody (2) it is an in vivo generated antibody (e.g., transgenic mouse system); (3) it binds to ErbB2 with an association constant of at least 10 12 M "1 ; (4) it binds to ErbB2 with an association constant of at least 10 11 M "1 ; (5) it binds to ErbB2 with an association constant of at least 10 10 M "1 ; (6) it binds to ErbB2 with an association constant of at least 10 9 M "1 ; (7) it binds to ErbB2 with an association constant of at least 10 6 M '1 ; (8) it binds to ErbB2 with a dissociation constant of 500 nM or less; (9) it binds to ErbB2 with a dissociation
  • the invention provides isolated nucleic acids encoding an anti-ErbB2 binding protein of the invention.
  • the nucleic acids may comprise DNA or RNA, and they may be synthetic (completely or partially) or recombinant (completely or partially).
  • Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T.
  • the invention also contemplates nucleic acids that comprise a coding sequence for a CDR1 , CDR2 or CDR3, a frame-work sequence (including FR1 , FR2, FR3 and/or FR4), a V H domain, a V L domain, or combinations thereof, as disclosed herein, or a sequence substantially identical thereto (e.g., a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical thereto, or that is capable of hybridizing under stringent conditions to the sequences disclosed).
  • the isolated nucleic acid has a nucleotide sequence encoding a heavy chain variable region and/or a light chain variable region of an anti-ErbB2 binding protein comprising at least one heavy chain CDR or light chain CDR, respectively, chosen from the CDR amino acid sequences found in SEQ ID Nos:1-95, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , 353, 355, 357, 359, 361
  • the nucleic acid encodes an anti-ErbB2 binding protein comprising one, two, or all 3 heavy chain CDRs, one, two or all 3 light chain CDRs or all 6 CDRS in any of an specifically exemplified antibody.
  • the nucleic acid can encode only the light chain or the heavy chain variable region, or can also encode an antibody light or heavy chain constant region, operatively linked to the corresponding variable region.
  • the light chain variable region is linked to a constant region chosen from a kappa or a lambda constant region.
  • the light chain constant region may also be a human kappa or lambda type.
  • the heavy chain variable region is linked to a heavy chain constant region of an antibody isotype chosen from IgG (e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 ), IgM, IgA 1 , IgA 2 , IgD, and IgE.
  • the heavy chain constant region may be an IgG (e.g., an IgG 1 ) isotype.
  • the nucleic acid compositions of the present invention while often in the native sequence (of cDNA or genomic DNA or mixtures thereof) except for modified restriction sites and the like, may be mutated in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired.
  • nucleotide sequences substantially identical to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
  • the nucleic acid differs (e.g., differs by substitution, insertion, or deletion) from that of the sequences provided (e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid).
  • ErbB2 binding proteins encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid specifically exemplified herein or to its complement. If necessary for this analysis the sequences should be aligned for maximum homology. "Looped out" sequences from deletions or insertions, or mismatches, are considered differences. The difference may be at a nucleotide(s) encoding a non-essential residue(s), or the difference may be a conservative substitution(s).
  • the invention also provides nucleic acid constructs in the form of plasmids, vectors, transcription or expression cassettes, that comprise at least one nucleic acid as described herein as well as a host cell that comprises at least one nucleic acid described herein.
  • Suitable host cells for the expression of a binding protein of the invention well be well known in the art and include mammalian, plant, insects, bacterial or yeast cells.
  • an anti-ErbB2 antibody of the invention that is encoded by the nucleic acid(s) comprising sequence described herein.
  • the method comprises culturing host cells under appropriate conditions to express the protein from the nucleic acid. Following expression and production, the encoded pp may be isolated and/or purified using any suitable technique, then used as appropriate.
  • the method can also include the steps of fusing a nucleic acid encoding a scFv with nucleic acids encoding a Fc portion of an antibody and expressing the fused nucleic acid in a cell.
  • the method can also include a step of germlining.
  • Antigen-binding fragments, V H and/or V L domains, and encoding nucleic acid molecules and vectors may be isolated and/or purified from their natural environment, in substantially pure or homogenous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the require function.
  • suitable host cells include mammalian cells, insect cells, plant cells, yeast cells, or prokaryotic cells, e.g., E. coli.
  • Mammalian cells available in the art for heterologous polypeptide expression include lymphocytic cell lines (e.g., NSD), HEK293 cells, Chinese hamster ovary (CHO) cells, COS cells, HeLa cells, baby hamster kidney cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.
  • lymphocytic cell lines e.g., NSD
  • HEK293 cells e.g., Chinese hamster ovary (CHO) cells
  • COS cells e.g., HeLa cells
  • baby hamster kidney cells e.g., baby hamster kidney cells
  • oocyte cells e.g., oocyte cells
  • all or a portion of an anti-ErbB2 antibody selected from S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1 R2C_CS_1 H 12,
  • S1R3A1_CS_15B8 S6R3_DP47_1A10, S6R2_DP47_1 E11 , S5R2_DP47_1H11, S6R3_CS_1 G5, S6R2_DP47_1 H11 , S5R3_DP47_1 A10, S5R2_DP47_1 D11 , S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1 H9 is expressed in HEK293 or CHO cells.
  • one or more nucleic acids encoding an anti-ErbB2 binding protein of the invention are placed under the control of a tissue-specific promoter (e.g., a mammary specific promoter) and the antibodies are produced in transgenic animals.
  • a tissue-specific promoter e.g., a mammary specific promoter
  • the antibodies are produced in transgenic animals.
  • the antibodies are secreted into the milk of the transgenic animal, such as a transgenic cow, pig, horse, sheep, goat or rodent.
  • Suitable vectors may be chosen or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences.
  • the vectors may also contain a plasmid or viral backbone.
  • a nucleic acid encoding all orjpart of an anti-ErbB2 binding protein of the invention may be introduced into a host cell by any readily available means.
  • suitable transfection techniques may include calcium phosphate, DE ⁇ AE-Dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses, e.g., vaccinia or baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.
  • DNA introduction may be followed by a selection method (e.g., drug resistance) to select cells that contain the nucleic acid.
  • Anti-ErbB2 binding proteins of the invention may be ErbB2 agonists or antagonists.
  • An agonist ErbB2 binder of the invention increases HER2 tyrosine phosphorylation in the absence or presence of other HER2 agonists such as Heregulin or Epidermal Growth Factor (EGF).
  • Certain HER2 agonists of the invention increase phosphorylation of HER2 pathway proteins.
  • the agonist of the invention increase phosphorylation of AKT, MAPK and/or ERK.
  • the HER2 agonist of the invention decreases proliferation and/or increases cell death of a cancer cell, in vitro and in vivo.
  • Anti-ErbB2 binding proteins that act as antagonists to ErbB2 can be used to reduce at least one ErbB2-mediated activity, such as reducing ErbB2-mediated tyrosine phosphorylation, decreased heterodimerization of ErbB2 with other ERBB-family members, decreased ErbB2-mediated cell signalling and decreased growth or proliferation of ErbB2- expressing cells.
  • anti-ErbB2 binding proteins of the invention are used in a method for decreasing tumor growth, the method comprising contacting an ErbB2 expressing cell with a binding protein of the invention to modulate cell proliferation, cytolytic activity, cytokine secretion, or chemokine secretion.
  • the binding proteins of the invention can be used to directly or indirectly inhibit or reduce the activity (e.g., proliferation, differentiation, and/or survival) of cells expressing ErbB2, and, thus, can be used to treat a variety of disorders including hyperproliferative disorders.
  • the binding proteins of the invention can be used to treat hyperproliferative disorders associated with activity of ErbB2 by administering the antibodies in an amount sufficient to inhibit or reduce hyperproliferation and/or to increase cell death, such as by apoplosis of ErbB2 expressing cells in a subject and allowing the antibodies to treat or prevent the disorder.
  • ErbB2 is expressed in a number of cancers including, but not limited to, breast, bladder, cervical, ovarian, prostate, testicular, oral, colorectal, lung and pancreatic, cancers and in childhood medulloblastoma, oral squamous cell carcinoma, gastric cancer cholangio carcinoma, osteosarcoma, primary Fallopian tube carcinoma, salivary gland tumors and synovial sarcoma.
  • Binding proteins of the invention may be used to inhibit the progression of neoplasms, e.g. squamous cell carcinomas, basal cell carcinomas, transitional cell papillomas and carcinomas, adenomas, adenocarcinoma.
  • an anti-ErbB2 binding protein of the invention can be administered to a subject in need thereof as part of a regimen that comprises another therapeutic modality, such as surgery or radiation.
  • a composition suitable for pharmaceutical use comprising at least one anti-ErbB2 binding protein further comprises at least one additional therapeutic agent.
  • the therapy is useful for treating ErbB2-mediated pathological conditions or disorders including cancer.
  • the term "in combination" in this context means that the binding protein composition and the additional therapeutic agent are given as part of a treatment regimen.
  • the anti-ErbB2 binding protein is administered substantially contemporaneously, either simultaneously or sequentially with another therapeutic agent, including one being a pretreatment in relation to the other.
  • the first of the two agents is still detectable at effective concentrations at the site of treatment.
  • the first of the two compounds is not detectable at effective concentrations at the site of treatment.
  • a treatment regimen may comprise two or more anti-ErbB2 antibodies of the invention.
  • the binding molecules may be ones that bind the same or nearby regions of HER2, as illustrated for example by blocking or cross-blocking each other's binding to HER2, or they may bind to different regions of HER2, as shown by lack of cross-blocking.
  • Two or more anti-ErbB2 binding molecules of the invention may be co-formulated, co-administered or merely be part of the same treatment regimen.
  • the combination therapy can include at least one anti-ErbB2 binding protein of the invention co-formulated with, co-administered with, or administered as part of the same therapeutic regimen as at least one additional therapeutic agent.
  • the additional agents may include at least but is not limited to mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics, antiproliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-l inhibitors, cox-ll inhibitors, radiation, cell cycle inhibitors, enzymes, anti-hormones, statins, and anti-androgens.
  • At least one anti-ErbB2 binding protein can be co- formulated with, and/or co-administered with, at least one anti-inflammatory drug, immunosuppressant, metabolic inhibitor, and enzymatic inhibitor.
  • an anti-ErbB2 antibody can be used in combination with at least one binding protein, such as an antibody, directed at other cancer targets.
  • Another aspect of the present invention accordingly relates to kits for carrying out the administration of the anti-ErbB2 binding protein alone or in combination with other therapeutic agents.
  • the kit comprises at least one anti-ErbB2 binding protein formulated in a pharmaceutical carrier, and at least one additional therapeutic agent, formulated as appropriate in one or more separate pharmaceutical preparations.
  • the present inventive binding proteins can be administered in combination with (e.g., prior to, concurrently with, or subsequent to) one or more other therapeutic agents.
  • Such therapeutic agents include, for example, cytotoxic agents that inhibit or prevent the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. 1131 , 1125, Y90 and Re186), chemotherapeutic agents, growth inhibitory agents, cytokine, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as
  • Poulenc Rorer Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • ifosfamide mitomycin C; mitox
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • a growth inhibitory agent when used herein refers to a compound or composition that inhibits growth of a cell, especially an ErbB2-overexpressing cancer cell either in vitro or in vivo.
  • the growth inhibitory agent can be one that significantly reduces the percentage of ErbB2 overexpressing cells in S phase and the binding proteins of the present invention may potentially sensitize the cells to such an S phase agent.
  • S-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo Il inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), include agents that induce G1 arrest and M-phase arrest. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1 , entitled “Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor, fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor
  • growth hormone such as
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • the invention also pertains to immunoconjugates comprising the binding proteins described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a radioconjugates are particularly indicated for those binding proteins of the invention that internalize in Her2 expressing cells, as shown in the Examples section.
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated anti-ErbB2 binding proteins. Examples include 212Bi, 1311, 131 In 1 90
  • Immunoconjugates comprising a member of the potent family of antibacterial and antitumor agents, known collectively as the calicheamicins or the LL-E33288 complex, (see U.S. Pat. No. 4,970,198 (1990)) are also contemplated.
  • the most potent of the calicheamicins is designated v 1 , which is herein referenced simply as gamma.
  • These compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides, at the same time introducing a functional group such as a hydrazide or other functional group that is useful in attaching a calicheamicin derivative to a carrier. (See U.S. Pat.
  • Conjugates of the binding protein and cytotoxic agent can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidy
  • a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987).
  • Carbon-14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the binding protein.
  • Effective amounts of the other therapeutic agents are well known to those skilled in the art. However, it is well within the skilled artisan's purview to determine the other therapeutic agent's optimal effective amount range.
  • the binding proteins of the present invention and the other therapeutic agent(s) can act additively or, alternatively, synergistically.
  • either the effective amount of the binding protein of the present invention or the other therapeutic agent(s) can be administered in an amount that is less than its effective amount would be where the other therapeutic agent is not administered. In this case, without being bound by theory, it is believed that the two (or more) act synergistically.
  • a binding protein of the invention may also be used to detect the presence of ErbB2 or ErbB2 expressing cells in a biological sample. By correlating the presence or level of ErbB2 with a medical condition, one of skill in the art can diagnose the associated medical condition, including cancer.
  • Binding protein-based including antibody-based detection methods are well known in the art, and include ELISA, radioimmunoassays, immunoblots, Western blots, flow cytometry, immunofluorescence, immunoprecipitation, and other related techniques.
  • the antibodies may be provided in a diagnostic kit that incorporates at least one of these procedures to detect ErbB2.
  • the kit may contain other components, packaging, instructions, or other material to aid the detection of the protein and use of the kit.
  • Binding proteins of the invention may be modified with detectable markers, including ligand groups (e.g., biotin), fluorophores and chromophores, radioisotopes, electron-dense reagents, or enzymes.
  • Enzymes are detected by their activity. For example, horseradish peroxidase is detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB tetramethylbenzidine
  • Other suitable binding partners include biotin and avidin, IgG and protein A 1 and other receptor-ligand pairs known in the art.
  • Binding proteins of the invention can also be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to at least one other molecular entity, such as another antibody (e.g., a bispecific or a multispecific antibody), toxins, radioisotopes, cytotoxic or cytostatic agents, among others for therapeutic use.
  • another antibody e.g., a bispecific or a multispecific antibody
  • toxins e.g., a bispecific or a multispecific antibody
  • cytotoxic or cytostatic agents e.g., cytotoxic or cytostatic agents
  • the anti-ERRB2 binding proteins can be used to detect the presence, isolate, and/or to quantitate ErbB2-expressing cells in a sample from a subject or by in vivo imaging.
  • compositions comprising an anti-ErbB2 binding protein of the invention.
  • the compositions may be suitable for pharmaceutical use and administration to patients.
  • the compositions comprise a binding protein of the present invention and a pharmaceutically acceptable carrier.
  • the composition may optionally comprise a pharmaceutical excipient.
  • pharmaceutical excipient includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Use of these agents for pharmaceutically active substances is well known in the art.
  • the compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. Pharmaceutical compositions may be topically or orally administered, or capable of transmission across mucous membranes. Examples of administration of a pharmaceutical composition include oral ingestion or inhalation. Administration may also be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, cutaneous, or transdermal.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include at least one of the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose.
  • a sterile diluent such as water, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • Solutions or suspensions used for intravenous administration include a carrier such as physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), ethanol, or polyol.
  • a carrier such as physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), ethanol, or polyol.
  • the composition must be sterile and fluid for easy syringability. Proper fluidity can often be obtained using lecithin or surfactants.
  • the composition must also be stable under the conditions of manufacture and storage. Prevention of microorganisms can be achieved with antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.
  • isotonic agents sucrose
  • polyalcohols mannitol and sorbitol
  • sodium chloride may be included in the composition.
  • Prolonged absorption of the composition can be accomplished by adding an agent that delays absorption, e.g., aluminum monostearate and gelatin.
  • compositions include an inert diluent or edible carrier.
  • the composition can be enclosed in gelatin or compressed into tablets.
  • the antibodies can be incorporated with excipients and placed in tablets, troches, or capsules.
  • Pharmaceutically compatible binding agents or adjuvant materials can be included in the composition.
  • the tablets, troches, and capsules may contain (1 ) a binder such as microcrystalline cellulose, gum tragacanth or gelatin; (2) an excipient such as starch or lactose, (3) a disintegrating agent such as alginic acid, Primogel, or corn starch; (4) a lubricant such as magnesium stearate; (5) a glidant such as colloidal silicon dioxide; or (6) a sweetening agent or a flavoring agent.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose
  • a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide
  • (6) a sweetening agent or a flavoring agent.
  • the composition may also be administered by a transmucosal or transdermal route.
  • antibodies that comprise a Fc portion may be capable of crossing mucous membranes in the intestine, mouth, or lungs (via Fc receptors).
  • Transmucosal administration can be accomplished through the use of lozenges, nasal sprays, inhalers, or suppositories.
  • Transdermal administration can also be accomplished through the use of a composition containing ointments, salves, gels, or creams known in the art.
  • penetrants appropriate to the barrier to be permeated are used.
  • the antibodies are delivered in an aerosol spray from a pressured container or dispenser, that contains a propellant (e.g., liquid or gas) or a nebulizer.
  • the binding proteins of this invention are prepared with carriers to protect against rapid elimination from the body.
  • Biodegradable polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid
  • Methods for the preparation of such formulations are known by those skilled in the art.
  • Liposomal suspensions can be used as pharmaceutically acceptable carriers too.
  • the liposomes can be prepared according to established methods known in the art (U.S. Patent No. 4,522,811 ).
  • the binding proteins or compositions of the invention are administered in therapeutically effective amounts as described. Therapeutically effective amounts may vary with the subject's age, condition, sex, and severity of medical condition. Appropriate dosage may be determined by a physician based on clinical indications.
  • the binding proteins or compositions may be given as a bolus dose to maximize the circulating levels of protein for the greatest length of time. Continuous infusion may also be used after the bolus dose.
  • the term "subject" is intended to include human and non- human animals. Subjects may include a human patient having a disorder characterized by cells that express ErbB2, e.g., a cancer cell or an immune cell.
  • non-human animals of the invention includes all vertebrates, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.
  • dosage ranges that can be administered to a subject can be chosen from: 1 ⁇ g/kg to 20 mg/kg, 1 ⁇ g/kg to 10 mg/kg, 1 ⁇ g/kg to 1 mg/kg, 10 ⁇ g/kg to 1 mg/kg, 10 ⁇ g/kg to 100 ⁇ g/kg, 100 ⁇ g/kg to 1 mg/kg, 250 ⁇ g/kg to 2 mg/kg, 250 ⁇ g/kg to 1 mg/kg, 500 ⁇ g/kg to 2 mg/kg, 500 ⁇ g/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 mg/kg , 10 mg/kg to 25 mg/kg, 15 mg/kg
  • dosages may be administered daily, weekly, biweekly, monthly, or less frequently, for example, biannually, depending on dosage, method of administration, disorder or symptom(s) to be treated, and individual subject characteristics. Dosages can also be administered via continuous infusion (such as through a pump). The administered dose may also depend on the route of administration. For example, subcutaneous administration may require a higher dosage than intravenous administration.
  • Dosage unit form refers to physically discrete units suited for the patient. Each dosage unit contains a predetermined quantity of antibody calculated to produce a therapeutic effect in association with the carrier. The dosage unit depends on the characteristics of the antibodies and the particular therapeutic effect to be achieved.
  • Toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 IED 50 .
  • Binding proteins that exhibit large therapeutic indices may be less toxic and/or more therapeutically effective.
  • the data obtained from the cell culture assays and animal studies can be used to formulate a dosage range in humans.
  • the dosage of these compounds may lie within the range of circulating antibody concentrations in the blood, that includes an ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage composition form employed and the route of administration.
  • the therapeutically effective dose can be estimated initially using cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of antibody that achieves a half-maximal inhibition of symptoms).
  • the effects of any particular dosage can be monitored by a suitable bioassay. Examples of suitable bioassays include DNA replication assays, transcription-based assays and ErbB2 binding assays.
  • Single chain fragment variable (scFv) moieties that bind to the extracellular domain (ECD) of Her2 (ErbB2) were identified following three rounds of selection using three phagemid libraries: the Bone Marrow Vaughan (BMV) library (Vaughan et al, 1996), the combined spleen (CS) library and the DP47 library (unpublished).
  • BMV Bone Marrow Vaughan
  • CS combined spleen
  • DP47 library unpublished.
  • Her2-Fc proteins or cell lines expressing various forms of Her2 were used during the selection and subsequent screening steps (see Table 3). The selection strategies are outlined in Figure 1.
  • phage and magnetic streptavidin beads were blocked separately in 3% milk/PBS for 1 hour at room temperature in a rotary mixer (20 rpm). Each selection was preceded by a de-selection step. For de-selection, blocked phage were incubated with the pre-blocked magnetic beads and incubated for one hour on a rotary shaker (20 rpm). The de-selected library was collected by pelleting the beads using a magnetic separator. A 1 ⁇ M concentration of a non-biotinylated competitor protein (eg, irrelevant MlgG2a protein) was added to the de-selected phage and incubated for a further hour.
  • a non-biotinylated competitor protein eg, irrelevant MlgG2a protein
  • Biotinylated selection antigen (at various concentrations as indicated in Figure 1 ) was incubated with the de-selected phage library for 2 hours at room temp on a rotary mixer (20 rpm) followed by a 15 minute incubation with pre-blocked magnetic beads. Beads were separated using a magnetic separator and washed 10 times with PBS/0.1% Tween 20 and 3 times with PBS. Bound phage were eluted by incubation with a 10 ug/ml solution of trypsin in PBS for 30 minutes at 37°C (100 rpm) followed by separation from the magnetic beads.
  • de-selection cells ie. cells not expressing the antigen of interest
  • 2 x 10 7 capture (i.e., selection) cells cells expressing the antigen of interest
  • PBS/5 mM EDTA washed twice with PBS.
  • Cells were blocked with 3% milk/1% BSA/PBS for 1 hour at 4°C on a rotary mixer (20 rpm).
  • De-selection cells were collected by centrifugation, re-suspended in blocked phage and incubated at 4°C as before.
  • Both the capture and de-selection cells were pelleted and the capture cells were resuspended with the de-selected phage supernatant and incubated at 4°C as before.
  • the capture cells were washed three times with cold PBS/0.1 % Tween 20 and three times with cold PBS. Phage were eluted by re-suspending the cells in a 10 ⁇ g/ml trypsin solution and incubated for 30 min at 37°C (100 rpm). Eluted phage were harvested in the supernatant following centrifugation of cells. Eluted phage were used to infect 10 ml of an E.
  • coli TG1 culture that had been grown to mid-logarithmic phase (corresponding to an OD 60O of ⁇ 0.5).
  • Bacteria were infected with phage for 1 hour at 37°C with shaking at 150 rpm, concentrated following a centrifugation step and plated on 2X TY agar bioassay plates containing 2% glucose and 100 ug/ml ampicillin (2X TYAG).
  • 2X TYAG 2X TYAG bioassay plates containing 2% glucose and 100 ug/ml ampicillin
  • Various dilutions of E. coli culture infected with either input or output phage were also plated on 2X TYAG agar to determine phage titers.
  • Example 2 Preparation of phage or crude periplasmic material for use in ELISAs
  • ScFvs can be expressed either on the surface of a phage particle or in solution in the bacterial periplasmic space, depending upon the growth conditions used.
  • 96-deepwell plates containing 2X TY media with 0.1 % glucose/100 ⁇ g/ml ampicillin were inoculated from thawed glycerol stocks (one clone per well) using the QPix2 Colony picker (Genetix) and grown at 37°C (999rpm) for ⁇ 4 hours. Cultures were induced with IPTG at a final concentration of 0.02 mM and grown overnight at 3O 0 C (999 rpm). The contents of the bacterial periplasm (peripreps) were released by osmotic shock. Briefly, plates were centrifuged and pellets were resuspended in 150 ⁇ l
  • HEPES periplasmic buffer 50 mM HEPES, pH7.4/0.5mM EDTA/20% Sucrose
  • 150 ⁇ l 1 :5 HEPES:water 150 ⁇ l 1 :5 HEPES:water and incubated on ice for 30 minutes. Plates were centrifuged and the scFv-containing supernatant was harvested.
  • phage expressing scFv on their surface, 96-well plates containing 150 ⁇ l 2X TY media with 2% glucose/100 ⁇ g/ml ampicillin were inoculated from thawed glycerol stocks as described above and grown at 37°C (700 rpm) for ⁇ 4 hours. 20 ⁇ l of a 1 :1000 dilution of helper phage (- 2 x 10 8 pfu) was added and the plates incubated for a further hour at 37°C (300 rpm).
  • kanamycin/non-glucose containing media 2X TY with 50 ⁇ g/ml kanamycin and 100 ug/ml ampicillin. Plates were grown overnight at 30 0 C (700 rpm) and phage were harvested in the supernatant following centrifugation.
  • ScFv's are: S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1 R2C_CS_1 H12, S1 R2A_CS_1 D3, S1 R3B2_BMV_1 E1 , S1 R3C1_CS_1 D3, S1 R3B2_DP47_1 E8, S1 R3B2_BMV_1 G2, S1R3B2_BMV_1 H5, S1 R3C1_CS_1A6, S1 R3B2_DP47_1C9, S1 R3B2_DP47_1 E10, and S1 R3C1_CS_1 B10 ( Figures 2 and 3).
  • Example 3 ELISA to test Her2 protein construct binding by scFvs expressed in the E. coli periplasm, on the surface of phage, or in mammalian cells as Fc fusions
  • Various Her2-Fc proteins e.g., HerOO ⁇ P, HerO17P, HerO18P, etc.
  • a negative control murine lgG2a protein were coated overnight at 4°C on 96-well Nunc Maxisorp at a concentration of 1 ug/ml in PBS.
  • pre-blocked streptavidin- coated plates were coated with biotinylated Her2-Fc proteins for 1 hour at room temperature at a concentration of 1 ug/ml in block buffer (3% skim milk/1% BSA/PBS). Plates were washed three times using PBS and blocked for 1 hour at room temperature in 3% skim milk/1 % BSA/PBS. Phage or peripreps were prepared as described above and were blocked for 1 hour at room temperature in an equal volume of 6% skim milk/1% BSA/PBS. Blocked plates were washed five times with PBS and 50 ⁇ l/well of blocked phage or periprep were transferred to the appropriate plates and incubated for 1 hour at room temperature.
  • HERCEPTIN® (trastuzumab) (in blocking buffer) was added to well H12 of each plate to serve as a positive control. Plates were washed five times with PBS prior to the addition of a 1 :250 dilution of anti-myc peroxidase (Roche), a 1 :2500 dilution of anti-M13 peroxidase (Amersham Biosciences) or a 1 :5000 or 1 :1000 dilution of goat anti-human peroxidase (Southern Biotech) secondary antibody to detect bound scFv, phage, HERCEPTIN® (trastuzumab) or SMIP, respectively.
  • SMIPs were used to capture 3-fold serial dilution (9-0 ⁇ g/ml) of soluble protein sample (see Figure 27). Captured soluble protein was detected using 0.1 mg/ml anti-c-Erb B2/c-Neu (Ab-5) mouse mAb (TA-1 ; binds ECD; Calbiochem) and detected using HRP-conjugated Goat anti-mouse IgG (Fcg Subclass 1 specific; Jackson ImmuonoResearch).
  • FIG. 6A-C The results of the SMIP binding assays are shown in Figure 6A-C, Figure 7A-7D, Figure 8, AND Figures 28-30.
  • Figure 8 the binding of HER018, HER026- HER039, and Herceptin® (trastuzumab) and HER018, to Her2 protein constructs was scored as -, +, ++ or +++; the, while the binding of HER071-HER087 to Her2 protein constructs was scored as a - or +.
  • Figure 28 the binding of HER SMIPs to Her2 protein constructs was scored as 0, +, ++, or +++, and cross-reactivity and binding domain are shown.
  • Figure 29 is a graphical summary of the results.
  • HER085 bound soluble full length Her2 ectodomain (ECD) (SIIS dimer) but not soluble Her2 EQR (SIIS lacking membrane proximal amino acids ASPLTSIIS). This indicated that HER085 binding domain required "stumpy" amino acids ASPLTSIIS.
  • Table 1 The results are summarized in the following Table.
  • Example 4 ELISA to measure binding of scFvs (expressed in the Periplasm or on the surface of phage) to Her2-expressed cells
  • Blocked plates were washed five times with PBS (+ Ca/Mg ions) and 50 ⁇ l/well of blocked phage or periprep were transferred to the appropriate plates and incubated for 1 hour at room temperature.
  • Each well of a 6 well plate was seeded with 2 x 10 5 cells and incubated overnight at 37 0 C / 5% CO 2 .
  • Cells were then treated with antibody or SMIP (at 10 ug/ml final) (in triplicate) and incubated for another 24 or 48 hours.
  • the cells were pulsed with 50 uM BrdU (Sigma) for 30 minutes at 37 0 C, the media was removed, and the cells were treated with trypsin (except Ramos) and then 3-3.5 x 10 5 cells per well were stained in 100 ⁇ l Staining Buffer in the presence or absence of a SMIP or antibody one of three different concentrations (ranging from 200 nM to 0.27 nM).
  • the SMIP or antibody treatment was removed and the cells were washed three times with PBS, pH 7.2-7 A with 0.1% TWEEN®-20 (PBS-T).
  • a secondary antibody (5 ug/ml Alexa Fluor 488-conjugated Goat anti-Human IgG; Molecular Probes #A-11013) was then added and incubated for 1-2 hours at room temperature. The secondary antibody was removed and the cells washed again three times with PBS-T. The cells were then fixed in 1% paraformaldehyde in Staining Buffer and analyzed 1 hour to 1 day later.
  • JIMT-1 ErbB2 epitopes may be partially blocked by MUC4 (Peter Nagy, Elza Friedlander, Minna Tanner, Anita I. Kapanen, Kermit L. Carraway, Jorma Isola, and Thomas M. Jovin.
  • the sensitivity may be due to trypsin cleavage of other molecules that are needed for the presentation/exposure of the "stumpy” peptide or the maintenance of Her2 p95 ("stumpy”) on the cell surface.
  • PCR amplification of scFvs was carried out using the KOD HOT START DNA Polymerase kit (Novagen) in accordance with the manufacturers instructions.
  • 0.2 ⁇ M each of the M13rev (5' G GAAACAG CTATG ACC ATG A 3') (SEQ ID NO: 247) forward and Mycseq (5' CTCTTCTGAGATGAG I I I I G 3') (SEQ ID NO: 248) reverse primers were used.
  • 5 ⁇ l of a 1:10 dilution of a stationary phase bacterial culture was used as the template for a final reaction volume of 20 ⁇ l.
  • the cycling conditions used were a 2 minute hot start at 94 0 C, 25 cycles of denaturation at 94 0 C (1 minute), primer annealing at 42 0 C (30 seconds) and extension at 72 0 C (1 min), followed by a final 5 minute extension at 72 0 C.
  • PCR products were verified by agarose gel electrophoresis and cleaned up with Exol/SAP (shrimp alkaline phosphatase) prior to sequencing of both strands with primers 145837 (5 1 GGAGATTTTCAACGTGAA 3') (SEQ ID NO: 249) and 142051 (5'
  • Binding of different Her2-directed binders (antibodies and SMIPs) to monomeric Her2 ECD and truncations of dimeric Her2 ECD were determined using a BIACORE® T100 instrument (GE Healthcare, Biacore, Piscataway, NJ). We conducted the binding experiments in both orientations, i.e., first using anti-HER2 SMIPS as ligands and then as analytes.
  • Her2-directed binders were captured on a chip by a monoclonal mouse anti-human Fc (GE healthcare), which was covalently conjugated to a carboxylmethyl dextran surface (CM4) via amines using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride and ⁇ /-hydroxysuccinimide. The unoccupied sites of the activated surface were blocked by ethanolamine.
  • the capturing antibody (referred to as anti hFc) binds to the C H 2 domain of IgG Fc of all sub-classes and showed no discernible dissociation from the captured her2-binders during the course of the assay.
  • Her2 binders were reproducibly captured every cycle with CV not exceeding 1%. The binding was performed at 25 0 C in 0.01 M HEPES pH 7.4, 0.15 M NaCI 1 0.005% v/v SURFACTANT P20. Signal associated with binding to the negative control was used to subtract for bulk refractive changes. The kinetic parameters and affinities were determined using BIAEVALUATION software. SMIPS as analvtes [0230] In these experiments, the trastuzumab (HERCEPTIN®) and and anti-HER2 SMIPs were used as the analytes and the soluble HER2 receptors were used as the ligands.
  • HERCEPTIN® trastuzumab
  • anti-HER2 SMIPs were used as the analytes and the soluble HER2 receptors were used as the ligands.
  • SMIPs and trastuzumab were flowed over a histidine-tagged monomeric HER2 receptor that was bound to a Ni 2+ -nitrilotriacetic acid surface.
  • SMIPs and trastuzumab were flowed over a histidine-tagged HER2 receptor that was captured by an anti-6-histidine-tagged monoclonal antibody conjugated to a CM4 surface.
  • SMIPs and trastuzumab were flowed over a HER2 receptor that was directly amine-coupled to a CM4 surface. The binding in each of these three experimental designs was performed at 25 0 C.
  • the HER067, HER033, HER030/HER094, HER 146, HER116 and HER102 SMIPS bound more strongly to dimeric soluble HER2 recpetor than to monomeric HER2 receptor.
  • the HER033 and HER067 SMIPs have the same amino acid sequence, but the difference between them is that the former is produced in HEK cells while the latter is produced in CHO cells. Binding by HER033 and HER067 SMIPs is substantially the same. HER030 appears to bind less strongly than HerO33/HerO67 to the dimers.
  • HER2 Specificity for dimeric HER2 may be advantageous in that such binders may have increased selectivity for tumors and may not bind, or show reduced binding to tissues that express low levels of HER2 and/or where ligand independent homodimer formation is limited.
  • Such HER2 binders with reduced binding to non-tumor target tissues e.g., cardiac tissues
  • a lack of binding to shed HER2 ectodomain would reduce the effective dose compared to a HER2-binding agent that has significant binding to shed ECD.
  • DELFIA Inducer (with Triton® X-100, glycine, HCI 1 and chelator) was then added to the cells (200 ⁇ l/well) and incubated with shaking for 15 minutes at RT. Fluorescence was measured using Flex Station® 3 in Time resolved fluorescence mode (Molecular Devices, Sunnyvale, CA).
  • MDA-MB-361 breast cancer cells were plated in 96-well format and treated with anti-Her2 or control reagents for indicated concentrations and times (24-96hr).
  • media DMEM plus 10% FBS
  • PBS phosphate-buffered saline
  • nuclei stained with DAPI (Molecular Probes). Stained nuclei were counted using Cellomics High Content assay measuring fluorescence at 36OnM. The results are shown in Figure 38.
  • apoptosis assay For apoptosis assay, fixed cells were permeabilized by treatment with 0.2% Triton 100 in PBS prior to primary staining with mouse anti-cleaved PARP antibody (Cell Signaling Technologies) and secondary staining with goat anti-mouse IgG labeled with ALEXA488 (Invitrogen). Fluorescence was measured in Cellomics High Content assay at 488nM.
  • ATP Lite First Step assay (Perkin Elmer) was used to assess cellular viability by measuring ATP levels via luminescence (ATP luciferase).
  • SMIPs were added to the cells at the desired concentration and then incubated at 37 0 C / 5% CO 2 for 4 (SKBR3, MDA-MB-453, MDA-MB- 361 , MDA-MB-175), 5 (BT474), or 7 (MDA-MB-361 ) days.
  • lyophilized ATP Lite substrate is reconstituted with 10 ml of ATP Lite substrate/lysis solution and allowed to sit at room temperature for 10 minutes. This reconstituted substrate solution was added to the cells (100 ⁇ l/well) and read luminescence on Top Count Reader (Packard).
  • the results of the proliferation assays are shown in Figures 10-12 and Figures 36-38 and are summarized in Figure 39.
  • the anti-HER2 SMIPS represent different groups of HER2 binders that bind different domains of HER2 and having differential ability to decrease proliferation in multiple cell lines.
  • anti- HER2 SMIPS reduce proliferation of a different repertoire of cell lines than HERCEPTIN®
  • the SMIP form of HERCEPTIN® has a different repertoire of cell killing than the parent antibody
  • HER2 SMIPS differ from each other in the cell lines in which they reduce proliferation.
  • the blocking solution was removed and primary antibody (in PBS with 3% horse serum or PBS with 1%BSA, and 0.1% Triton® X-100) was added for 1 hour at room temperature (or overnight at 4 0 C).
  • the primary antibodies used (at 0.125 ⁇ g/well) were (1 ) rabbit anti-phospho-akt (Ser473) (Cell Signaling, Danvers, MA); (2) mouse anti-phospho- Erk1/2 (Cell Signaling, Danvers, MA); and (3) rabbit anti-phospho-ErbB2 (Abgent, San Diego, CA).
  • the primary antibody was removed and the cells were washed 3 times with PBS.
  • the secondary antibody in PBS with 3% horse serum or PBS with 1% BSA, and 0.1% Triton® X-100 was then added for 1 hour at room temperature (or overnight at 4 0 C) protected from light.
  • the secondary antibodies used at 0.2 ⁇ g/well were Alexa 488 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA) and DyLight 649 goat anti-ms IgG (Pierce, Rockford, IL). The secondary antibody was removed and the cells were washed 3 times with PBS.
  • MDA-MB-361 breast cancer cells were plated in 6-well plate to 80-90% confluency (DMEM plus 10% FBS) and treated with anti-Her2 or control reagents for 24hr with and without pretreatment with Heregulin (HRG - 15 min.) or EGF (30 min.).
  • Heregulin HRG - 15 min.
  • EGF EGF
  • Western blot analysis used either rabbit anti-Her2 antibody (Cell Signaling Technologies), anti-pHer2_Y1248 (Upstate) or anti-Actin (Santa Cruz) as primary antibody and subsequently stained with HRP-conjugated anti-rabbit IgG. Peroxidase activity was measured using ECLplus2 kit (GE Healthcare) following manufacturer's protocols and exposed to film. As shown in Figure 13, HER033 induces HER2 phosphorylation.
  • MDA-MB-361 breast cancer cells were plated in 96-well format and treated with anti-Her2 or control reagents for the concentrations and times (10min to 24hr) shown in Figure 15. Media was removed, cells washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton 100/PBS. Cells were subsequently stained with either rabbit anti-pAKT (Cell Signaling Technologies), anti-pERK (Cellomics), anti-pS6K (Cell Signaling Technologies), or anti-p38MAPK (Cell Signaling Technologies). Following PBS wash (3X), cells were stained with secondary goat anti-rabbit IgG antibody labeled with ALEXA594. Cell fluorescence was quantified using Cellomics High Content assay at 594nM.
  • HerO67 (HerO33) has agonistic activity (increased signaling) compared to trastuzumab (see Table 6). Moreover, HerO67 and HerO18 are generally a stronger inducer of Her2, Erk1/2, and Akt phosphorylation than trastuzumab. The increase was statistically significant as compared to the mock treatment when measured by the pairwise student T- test ( ⁇ 0.001 ). Table 6. Induction of phosphorylation by HER018, HER067, Herceptin and Heregulin
  • Her146 mediated antiproliferative activity is demonstrated by decrease in viable cell count in absence of co- treatment with the kinase inhibitor.
  • Inhibition of MEK with small molecule kinase inhibitor CL- 1040 between 0.4 and 3.7uM demonstrate dose dependent reversal of the Her146 mediated anti-proliferative activity, demonstrating that Her146 activity is mediated by hyperactivation of MEK kinase pathway activity.
  • Higher doses of CL-1040 inhibited cell proliferation by complete inhibition of MEK kinase activity.
  • siRNA against ERK1 or ERK2 was used to investigate the effect on SMIP anti-proliferative activity in MDA-MB-361 breast cancer cells. Briefly, the cells were reversed transfected with siRNA oligos (25nM) targeting ERK1 or ERK2 kinases, or with non-targeting control oligo (NTO) using Dharmafect 4 lipid and following manufacture's recommended protocols in 96-well plate format. Cells were grown 60hr in DMEM media plus 10% FBS and then treated with either HeM 46 (0.3ug/ml) or vehicle control as indicated.
  • siRNA oligos 25nM
  • NTO non-targeting control oligo
  • MDA-MB-361 breast cancer cells were grown in DMEM media supplemented with 10% FBS.
  • Cells were treated with either anti-Her2 SMIPs (HerO33, Her146), Herceptin or controls anti-CD20 SMIP or untreated.
  • cell populations were either treated with heregulin (HRG1 ), the ligand activator of Her3, or vehicle for a total of 24 or 48hr.
  • HRG1 heregulin
  • Cells were harvested and protein lysates size fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Protein blots were probed with anti-pHer2 (Upstate), anti-pHer3 (Cell Signaling Technologies) or anti-Actin (Santa Cruz, loading control) monoclonal antibodies.
  • each well of a 6 well plate was seeded with 2 x 10 5 cells (SKBR3 or BT474 (sensitive) or MDA-MB-453 or MDA-MB-361 (resistant)) and incubated overnight at 37 0 C / 5% CO 2 .
  • Cells were then treated with antibody or SMIP (at 10 ⁇ g/ml final) (in triplicate) and incubated for another 24 or 48 hours.
  • the cells were pulsed with 50 uM BrdU (Sigma) for 30 minutes at 37 0 C, the media was removed, and the cells were treated with trypsin and harvested in a FACS tube on ice.
  • the cells were washed with PBS, fixed with 70% cold ethanol, and incubated on ice for 30 minutes. The ethanol was removed and then 2N HCI/0.5%Triton X-100 was added, and the cells were incubated for 30 minutes at room temperature (RT).
  • the acid was removed and neutralized with 0.1 M Na 2 B 4 O 7 for 15 min at RT.
  • FITC labeled anti-BrdU antibody was added (BD Bioscience) in PBS/0.5% TWEEN® 20/1% BSA, and the cells were incubated for 30 minutes at RT in the dark.
  • the FITC dye was removed, the cells washed, and then DAPI nuclear stain (Invitrogen) and RNAse A (Qiagen) each at 1 :1000 dilution was added and the cells were incubated 15 minutes in the dark and then analyzed by FACS.
  • Statistical analysis of the data was performed using ANOVA and Student's t-test.
  • HER116 appeared to behave a little differently than HER030/094, HER033/067, and HER146.
  • HER067, HER146, HER102, HER122 and Heregulin treated BT474 cells We also observed an increased number of cells in the G1 phase in Herceptin® treated SKBR3 and BT474 cells; HER033, HER067, HER146, and HER116 treated MDA-MB-453 cells at 24 hours; HER033, HER067, and HER146 treated MDA-MB-361 (JL) cells at 24 hours; HER094, HER067, and HER146 treated MDA-MB-361 (JL) cells at 48 hours; Herceptin treated MDA- MB-361 (ATCC) cells at 24 hours; and HER094, HER067, and HER146 treated MDA-MB- 361 (ATCC) cells at 48 hours.
  • Treatment with HER094, HER0333, HER067, HER146, HER116, HER124, and Heregulin resulted in an increase in the number of SKBR3 cells in S-phase at 24 hours.
  • HER124, and heregulin increased the number of BT474 cells in S-phase at 24 hours.
  • HER018, HER094, HER033, HER146, HER116, HER102, and heregulin decreased the number of BT474 in G2M phase.
  • MDA-MB-361 (ATCC) cells at 48 hours showed significantly decreased G2M phase cells following SMIP treatment (HER094, HER067, HER146 and heregulin).
  • mice were monitored (i.e., weighed and tumors measured) two to three times weekly. Mice were sacrificed if ulceration of tumor occurred, extreme body weight loss (greater than or equal 20%), tumor exceeded about 1200 to about 1500 mm 3 , or tumor inhibited mobility of a mouse. The study is continued for a total of about 60 days.
  • Treatment Mice were sorted into three groups of 11 mice each. Treatment began on day 0 (about six days after cell implantation).
  • mice of a group received intraperitoneal treatments twice a week (for a total of five treatments), which were given in equimolar amounts (900 nM) of (1) SMIP HER067 (100 ⁇ g), (2) Herceptin (136 ⁇ g, positive control), or (3) human IgG (136 ⁇ g, negative control). Survival and tumor size was recorded two to three times weekly. Results were graphed (+/- SEM) and analyzed using Prism software (see Figures 21 and 22).
  • mice were sorted into 4 groups: (1 ) HER146 (100 ⁇ g), (2) HER116 (100 ⁇ g), (3) Herceptin (136 ⁇ g, positive control) and (4) human IgG (136 ⁇ g, negative control). Survival and tumor size was recorded two to three times a week. Results were graphed (+/- SEM) and analyzed using Prism software (see Figures 46 and 47)
  • mice Male BALB/c nu/nu (nude) mice (18-23 g) and female nu/nu (nude) mice (18-23 g) were obtained from Charles River Laboratories, Wilmington, MA.
  • mice Female, athymic nude mice were exposed to total body irradiation (400 rads) to further suppress their residual immune system and facilitate the establishment of xenografts. Three days later, the irradiated mice were injected subcutaneously (SC) with 1x10 7 MDA-MB-361 cells in Matrigel (Collaborative Biomedical Products, Belford, MA, diluted 1 :1 in culture medium) in the dorsal, right flank. When the tumors reached the mass of 0.1 to 0.25 g, the tumors were staged to ensure uniformity of the treatment groups. Male, athymic Balb/c nude mice were injected s.c. with 1x10 7 cells in the right flank.
  • SC subcutaneously
  • Matrigel Collaborative Biomedical Products, Belford, MA, diluted 1 :1 in culture medium
  • Example 11 Identification and screening of antibodies that bind to the membrane proximal region of Her2/ERBB2
  • Ligand binding triggers ERBB2 dimerization and the activation of the intracellular kinase domain of ERBB2.
  • Autophosphorylation of C-terminal tyrosines triggers the recruitment to these sites of intracellular signal transducers that regulate cellular processes such as proliferation, differentiation, motility, adhesion, protection from apoptosis, and transformation.
  • ERBB2 is frequently over-expressed in breast cancer.
  • the existence of high levels of circulating soluble ERBB2 extracellular domain is associated with poor prognosis and decreased responsiveness to chemotherapy and endocrine therapy.
  • soluble ERBB2 extracellular domain arises by proteolytic cleavage of the extracellular domain of ERBB2.
  • the cleavage of the extracellular domain results in a truncated, cell-associated, ERBB2 fragment that contains the intracellular kinase domain and a potentially surface-exposed N-terminal membrane proximal sequence, EQRASPLTSIIS (amino acid residues 645-656 of HER2).
  • ERBB2 p95 This membrane-bound fragment (designated as ERBB2 p95 because of its molecular weight) shows potentially enhanced signalling activity. It has been speculated that the adverse prognosis observed in patients with high levels of ECD/ERBB2 may be related, at least in part, to an increase of truncated, signalling- competent, ERBB2 p95. [0265] Because the N-terminal membrane proximal sequence, EQRASPLTSIIS
  • stumpy region potentially remains on cell surface after the proteolytic cleavage of the extracellular domain, the stumpy region is a potential target for therapeutic intervention.
  • Herceptin® Trastuzumab
  • doxorubicin doxorubicin
  • cyclophosphamide cyclophosphamide
  • paclitaxel paclitaxel
  • Herceptin does not bind to the stumpy region of ERBB2.
  • an antibody that bind to the stumpy region of ERBB2 would be a more potent and effective inhibitor of the truncated, signalling-competent, ERBB2 p95.
  • scFv Single chain fragment variable moieties that bind to the membrane- proximal region of Her2 (ErbB2) that remains on the cell surface following cleavage and release of the soluble extra-cellular domain were identified following three rounds of selection using the Cambridge Antibody Technology (CAT) phage display libraries. Selection strategies are outlined in Figure 5. Three CAT libraries were used; the Bone Marrow Vaughan (BMV) library (Vaughan et al, 1996), the combined spleen (CS) library and the DP47 library (unpublished).
  • BMV Bone Marrow Vaughan
  • CS combined spleen
  • aliquots of phage and magnetic streptavidin beads (Dynabeads M-280 streptavidin) were blocked separately in 3% milk/PBS for 1 hour at room temperature in a rotary mixer (20 rpm). Blocked phage were incubated with a 100 nM concentration of the scrambled de-selection peptide in round 1 (the amount of de-selection peptide decreased in subsequent rounds as the concentration of the selection peptide decreased), incubated at room temperature for 1 hour on a rotary shaker (20 rpm), mixed with blocked magnetic beads and incubated for a further hour. The de-selected library was collected by pelleting the beads using a magnetic separator.
  • Biotinylated selection peptide (at various concentrations as indicated in Figure 5A) was incubated with the de-selected phage library for 2 hours at room temp on a rotary mixer (20 rpm) followed by a 15 minute incubation with pre-blocked magnetic beads. Beads were separated using a magnetic separator and washed 10 times with PBS/0.1% Tween 20 and 3 times with PBS. Bound phage were eluted by incubation with a 10 ug/ml solution of trypsin in PBS for 30 minutes at 37°C (100 rpm) followed by separation from the magnetic beads. [0267] Eluted phage were used to infect 10 ml of an E.
  • coli TG 1 culture that had been grown to mid-logarithmic phase (corresponding to an OD 6 oo of ⁇ 0.5).
  • Bacteria were infected with phage for 1 hour at 37°C with shaking at 150 rpm, concentrated following a centrifugation step and plated on 2X TY agar bioassay plates containing 2% glucose and 100 ug/ml ampicillin (2X TYAG).
  • 2X TYAG 2X TYAG bioassay plates containing 2% glucose and 100 ug/ml ampicillin
  • Various dilutions of E. coli culture infected with either input or output phage were also plated on 2X TYAG agar to determine phage titers.
  • Example 12 ELISA to measure binding of scFvs expressed in the periplasm or purified to biotinylated Her2 protein constructs
  • a streptavidin-coated 96 well plate (Greiner) was washed three times with PBS/0.05% Tween 20 and blocked for 1 hour at room temperature in 3% skim milk/1% BSA/PBS. Plates were washed three times with PBS/0.05% prior to the addition of a 1 mg/ml solution of biotinylated Her2-Fc proteins (HerOO ⁇ P, HerO17P, HerO18P, HerO54P) or a biotinylated negative control murine lgG2a protein. Plates were incubated for one hour at room temperature.
  • Peripreps were prepared as described in an earlier section and were blocked for 1 hour at room temperature in an equal volume of 6% skim milk/1% BSA/PBS. Blocked plates were washed five times with PBS/0.05% Tween 20 and 50 ml/well of blocked periprep (or purified scFv diluted in block buffer) were transferred to the appropriate plates and incubated for 1 hour at room temperature. A 1 ug/ml solution of Herceptin (in blocking buffer) was added to well H12 of each plate to serve as a positive control.
  • Heavy and light chain V regions from scFv clones are amplified with clone- specific primers. PCR products are digested with appropriate restriction enzymes and subcloned into vectors containing human IgGI heavy chain constant domain (for V H domains) or vectors containing human lambda or kappa light chain constant domains as appropriate (V L domains). The closest human germlines of the V H and V L segments are determined and this information is used to indicate whether kappa or lambda light chain constant domains are used. Correct insertion of V region domains into plasmids is verified by sequencing of plasmid DNA from individual E. coli colonies. Plasmids are prepared from E. coli cultures by standard techniques and heavy and light chain constructs are co-transfected into COS cells using standard techniques. Secreted IgG is purified using protein A sepharose (Pharmacia) and buffer exchanged into PBS.
  • Her102, Her116, HeM 46 or HerO18 or molar equivalent of antibody (HERCEPTIN® as a positive control, or Retuxan as a negative control).
  • HERCEPTIN® as a positive control
  • Retuxan as a negative control
  • the table below shows the SMIPs and antibodies that were used.
  • cells were also treated with either 1 nM pervanedate to increase ectodomain shedding, or 5ug/ml TIMP1 a protease inhibitor that results in blockage of Her2 cleavage.
  • Figures 5OA and 50B 1 SMIPs decrease shedding of the Her2 ectodomain. As shown in
  • SMIPs the mechanism for SMIPs' decreasing cell surface Her2 and shedding Her2 ectodomain may be that teh SMIP blocks Her2 cleavage, thus reducing shed ectodomain and production of p95 Her2.
  • SMIPs could increase Her2 internalization, thus reducing cell surface ECD. Similar mechanisms have been described for HERCEPTIN®.
  • SMIPs were labeled with FMAT Blue as per manufacturers directions (Applied Biosystems). Unlabeled competitor SMIPs or Antibodies were diluted to 40OnM in FMAT Blocking Buffer (44 ug/mL for SMIPs; 59.2 ug/mL for antibodies). Each protein was titrated 1 :3 in FMAt blocking buffer in duplicate V-bottom tissue culture 96-well plates in a final volume of 60ul/well. Cells (SKBR3) were added in 6OuI FMAT blocking buffer to give 36,000 cells/well.
  • Plates were incubated for 1 hour at room temperature before adding FMAT Blue labeled antibodies at a concentration determined to give maximal staining in the absence of competing unlabeled SMIP or antibody (5 ug/mL for HERCEPTIN®; 2ug/ml_ for HER018, 10ug/ml_ for all other HER SMIPs 1 and 2ug/ml for Rituxan and 2LM20-4 (anti-CD20 SMIP)). Plates were incubated at room temperateure for 45-60 minutes (10 minutes for Herceptin). Cells were spun down at 1250 rpm for 5 minutes and non-bound SMIPs and antibodies flicked off.
  • HERCEPTIN® binding is blocked by HERCEPTIN® and HER018 at low concentrations; HER067, HER102, HER146 at higher concentrations; and HER094 at very high concentrations.
  • HER018 binding is blocked by HER018 and HERCEPTIN® at low concentrations; HER067, HER102, HER146 at higher concentrations; and HER094 at very high concentrations.
  • HER067 binding is blocked by HERCEPTIN® and HER018 at low concentrations; HER067 and HER102 at higher concentrations; and HER094 and HER146 at very high concentrations.
  • HER067 binding is greatly enhanced by HER116 binding.
  • HER094 binding is blocked by HERCEPTIN® and HER018 at low concentrations; HER067 and HER102 at higher concentrations; and HER094 and HER146 at very high concentrations. Also, HER094 binding is greatly enhanced by HER1 16 binding.
  • HER102 binding is blocked by HERCEPTIN® and HER018 at low concentrations; and HER146 and HER102 at higher concentrations. Also, HER102 binding may be slightly enhanced with HER1 16 binding.
  • HER1 16 binding is blocked by HER116 at low concentrations. No other SMIPs or antibodies blocked HER116 binding.
  • HER146 binding is blocked by HERCEPTIN®, HER018 and HER102 at low concentrations; and HER146 at higher concentrations.
  • Anti-CD20 Ab and SMIP binding is not blocked by any HER2 SMIPs or antibodies.
  • the SMIP cross-blocking results are summarized in Figure 52.
  • Her2 SMIPs that do not cross-block each other have the potential to simultaneously bind to a Her2 molecule. Accordingly, there may be an additive mechanism of action for Her2 binding with SMIPs and antibodies. Further, there is a possibility for a combination treatment with multiple SMIPs or with a combination of SMIP and antibody. SMIPs could also be potential partners for bispecific molecules such as ScorpionsTM .
  • Hum-ZAP Advanced targeting Systems
  • SMIP single-chain antigen
  • Hum-ZAP is a saporin-conjugated anti- human Ig that targets and eliminates cells using the internalization of an antibody or SMIP.
  • a human IgG containing molecule such as a SMIP or antibody, that recognizes an extracellular domain of a cell surface antigen
  • Hum-ZAP is taken inside the cell by antibody or SMIP-mediated internalization.
  • the entrance of saporin into the cell will result in protein synthesis inhibition and eventual cell death after 2-4 days.
  • Cells in 90 ⁇ l of media were added to 96-well plates and incubated overnight. The following day, cells were treated by either: a) the addition of 5 ⁇ l of a SMIP and media; b) the addition of 5 ⁇ l goat IgG-SAP (goat anti- human IgG negative control) and media; or c) the addition of 5 ⁇ l of a SMIP and Hum-ZAP (Saporin-conjugated goat anti-human IgG). Cells were incubated a further 96 hours before being assayed for proliferation using standard BrdU-incorporation and Hoechst nuclear staining. Internalization was observed as a reduction in cell proliferation an plotted as percent of untreated control.
  • MDA-MB-361 (ATCC) cells This may be due to the fact that MDA-MB-361 (ATCC) cells grow slowly. Thus, longer treatment times with increased cell numbers may be necessary in order to detect a response.
  • MDA-MB-361 cells were grown in 96-well plate format and treated with anti-
  • Her2 SMIPs 1 Herceptin (Here) or control anti-CD20 SMIP for indicated times. Media was removed and cells fixed (4% paraformaldehyde) and permeabilized (0.2% TritonlOO). Cell surface or intracellular SMIPs or monoclonal antibodies were detected by staining with FITC- labeled anti-hulgG-Fc (see Figure 53A-F, panels A and B or with rabbit anti-Her2 mAB (Cell Signaling Technologies) with secondary FITC-labeled Goat-anti-Rabbit IgG (Molecular
  • Her116 demonstrated rapid binding and internalization of SMIP (Panel A:10min; Panel B: 1hr) and cell surface Her2 (Panel C: 1 hr) similar to Herceptin mAB.
  • Her146 treatment demonstrated slower kinetics of cell surface binding that was sustained for longer time periods (Panel B: 1hr) and confirmed with anti-Her2 cell surface localization (Panel C: 1hr).
  • Control anti-CD20 SMIP did not display binding at any time point as anticipated.
  • SMIPs and antibodies were labeled with CypHer ⁇ E (GE Healthcare) as per manufacturers direction. CypHer ⁇ E has little or no fluorescence at physiological pH, but fluoresces at low pH (e.g., when internalized into lysosomal compartments). Cells were plated in serum-free media and placed on ice for 5-10 minutes. Cells were then washed (1x) with cold media containing 1% FBS. Dilutions of CypHer ⁇ E labeled SMIPs or antibodies in ice cold serum-free media were added to cells and incubated on ice for 45 minutes. Cells were washed (1x) with ice cold media containing 1% FBS.
  • CypHer ⁇ E is imaged in the red channel (650-700nm), Hoechst in the blue channel (387-525nm) and the Alex-488 secondary antibody in the green channel (485-525nm).
  • HER018 and HER116 were rapidly internalized- within 10 minutes.
  • the presence of SMIP binding was confirmed after fixation with an anti-human Fc secondary Ab.
  • HER067, HER146, HER156, and HER169 were internalized more slowly. We observed some internalization of these SMIPs by 4 hours.
  • Herceptin was internalized at a faster rate in SMIP format (HER018) than as Ab.
  • SKBR3 cells were harvested with trypsin and washed.
  • Cells were labeled with BADTA (Perkin Elmer) by incubating 2x10 6 cells in 2ml media with 20 ⁇ l BADTA mix (5 ⁇ l BADTA reagent, 2 ⁇ l PF127, 13 ⁇ l DMSO) for 20 minutes at 37C.
  • Labeled cells were washed with PBS (4x) and resuspended in media at 400,000 cells/ml.
  • Cells (20,000 cells in 50 ⁇ l) were aliquoted into a V-bottom plate and 50 ⁇ l of 2x SMIP or antibody were added.
  • HER116m HER033/067 and HER094 have good to moderate FcDCC activity that is comparable to that of HERCEPTIN® and HER018.
  • SMIPs Stability of SMIPs in mouse plasma was determined by incubating SMIPs (200ug/ml) in mouse plasma or PBS at 37C or 4C for up to 96 hours, with samples removed at intermediate times. A dilution series was made for each SMIP sample and the concentration was determined by ELISA using plates coated with a Her2 ECD murine Fc fusion protein (Her2SIIS::muFc). Captured SMIP was detected using a HRP-conjugated secondary anti mouse Fc secondary antibody. Mouse plasma alone or an anti-CD20 antibody, were used as negative controls in these experiments.
  • SMIP/receptor mixture were subjected to size-exclusion chromatography combined with refractive index, multiple angle laser light scattering (SEC-RI-MALLS), using a TOSOH TSK G4000 SW ⁇ L column.
  • SEC-RI-MALLS multiple angle laser light scattering
  • TOSOH TSK G4000 SW ⁇ L column The mass of the resolved peaks was analyzed using ASTRA software (Wyatt Technology Corporation, CA). The results of the mass analysis are shown in Figure 58.
  • Cells were treated with the combination of SMIP/antibody and therapeutic an additional 24 hours before the cells were quantitated by counting cells using the nuclear stain, Hoechst, or by the ability of live cells to incorporate BrdU using standard assays.
  • a 5- fold dilution series was run for each assay/treatment with a maximal concentration of SMIP of 182nM and 10OuM Cisplatin, 10OnM Taxol, 100OnM Doxorubicin, or 10OnM Gemcitabine with the ratio remaining constant for each dilution.
  • the combination of SMIP and chemotherapy was compared to either SMIP or chemotherapy alone. Dose response curves of cells pre-treated with HER146 and then treated with various chemotherapeutic agents or combinations thereof are shown in Figure 59A-D.
  • Her2 SMIPs could have additive effects when administered with chemotherapeutic agents.
  • MDA-MB-453 cells treated with HER146 were more sensitive to chemotherapeutic agents (e.g., Cisplatin, Taxol, and Doxorubicin).
  • MDA- MB-361-JL cells treated with Her146 were more sensitive to some chemotherapeutic agents (e.g., Cisplatin, Taxol, and Gemcitabine) but not others (e.g., Doxorubicin).
  • Her2 was immunoprecipitated from 1mg RIPA lysate or 2mg Nonidet P-40 lysate using 5ug of SMIPs 1 5ug human IgG (as negative control) or 2ug mouse monoclonal antibody, 3B5, against the intracellular region of Her2 (positive control), lmmunoprecipitated protein is pulled down with protein A or protein G beads, washed and separated by SDS- PAGE.
  • the secondary antibody we used was 3 x 1 :5000 IRDye 800CW Donkey anti-Rabbit lgG(H+L) (LI-COR #926-32213, lot B70416-01 ). The results are presented in Figure 61. [0301] We found that HER156 and HER169 are capable of binding full-length
  • HER156 and HER169 could bind Her2 p95 ("Stumpy;" cleaved ErbB2 that should run at 95 KDa). For example, it was not clear to us whether p95 can be immunoprecipitated at detectable levels from SKBGR3 cells by either HER156 or HER169. It was possible that there was too little p95 in SKBR3 cells for detection.

Abstract

The present application provides novel binding proteins, including human binding proteins that specifically bind to the human ErbB2.

Description

THERAPEUTIC COMPOSITIONS AND METHODS
Cross Reference to Related Applications
[0001] This application claims the benefit of U.S. Provisional Applications 61/000,511 , filed October 25, 2007 and 61/062,433, filed January 24, 2008, and of PCT/US2008/006905 and Unites States application 12/156,159, both filed May 29, 2008.
Field of the Invention
[0002] This invention relates to binding proteins that bind erythroblastic leukemia viral oncogene homolog 2 (ErbB2), in particular, human ErbB2 (also known as HER2), and their use in regulating ErbB2-associated activities. The binding proteins disclosed herein are useful in diagnosing, preventing, and/or treating ErbB2 associated disorders, e.g., hyperproliferative disorders, including cancer, and autoimmune disorders, including arthritis.
Background of the Invention
[0003] The ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR or ErbB1 ), HER2 (ErbB2 or p185neu), . HER3 (ErbB3) and HER4 (ErbB4 or tyro2). Structurally, the ErbB receptors possess an extracellular domain (with four subdomains, I — IV), a single hydrophobic transmembrane domain, and (except for HER3) a highly conserved tyrosine kinase domain. Crystal structures of EGFR reveal a receptor that adopts one of two conformations. In the "closed" conformation, EGFR is not bound by ligand and the extracellular subdomains Il and IV remain tightly apposed, preventing inter-receptor interactions. Ligand binding prompts the receptor to adopt an "open" conformation, in which the EGFR receptor is poised to make inter-receptor interactions. [0004] The ErbB receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of ErbB ligands. EGFR is bound by at least six different ligands; epidermal growth factor (EGF)1 transforming growth factor alpha (TGF-α), amphiregulin, heparin binding epidermal growth factor (HB-EGF), betacellulin and epiregulin. A family of heregulin proteins resulting from alternative splicing of a single gene are ligands for ErbB3 and ErbB4. The heregulin family includes alpha, beta and gamma heregulins, neu differentiation factors (NDFs), glial growth factors (GGFs); acetylcholine receptor inducing activity (ARIA); and sensory and motor neuron derived factor (SMDF). [0005] HER2 was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis. Overexpression of ErbB2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
[0006] HER2 has been suggested to be a ligand orphan receptor. Ligand- dependent heterodimerization between HER2 and another HER family member, HER1 , HER3 or HER4, activates the HER2 signaling pathway. The intracellular signaling pathway of HER2 is thought to involve ras-MAPK and PI3K pathways, as well as MAPK-independent S6 kinase and phospholipase C-gamma signaling pathways. HER2 signaling also effects proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and an antiangiogenic factor, thrombospondin-1 (TSP-1). [0007] The full-length ErbB2 receptor undergoes proteolytic cleavage releasing its extracellular domain (ECD), which can be detected in cell culture medium and in patient's sera. The truncated ErbB2 receptor (p95ErbB2) that remains after proteolytic cleavage exhibits increased autokinase activity and transforming efficiency compared with the full- length receptor, implicating the ErbB2 ECD as a negative regulator of ErbB2 kinase and oncogenic activity.
[0008] A recombinant humanized version of the murine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821 ,337) is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® reportedly targets the C-terminal region of domain IV of ErbB2. HERCEPTIN® clinical activity is predominately dependent on antibody dependent cell mediated cytotoxicity (ADCC). Studies have suggested that HERCEPTIN® acts by triggering G1 cell cycle arrest.
[0009] Presently ErbB-directed therapeutics do not meet the current medical needs. ErbB-directed therapeutics have had only modest anti-tumor efficacy and are not as potent as anticipated from preclinical models. In most patients who initially respond to HERCEPTIN®, disease progression is noted within 1 year. In the metastatic setting, a median duration of roughly nine months was reported, at which point it appears that patients frequently become refractory to therapy. Studies have suggested that more complete blockade of the ErbB receptor family would be beneficial. As there are multiple functional domains of HER2, agents targeted to each of the domains could be a potentially valuable therapeutic. Additionally, there are harmful side effects of HERCEPTIN® treatment. Cardiac dysfunction, quantitated as a decrease in left ventricular ejection fraction (LVEF) of 10% from baseline or less than 50% total, was identified in roughly 7.1% of patients receiving HERCEPTIN® for 1 year versus 2.2% in patients randomized to observation in the HERA trial. Rates of severe and symptomatic congestive heart failure (CHF) were also significantly higher in the group randomized to HERCEPTIN®. Potentially, agents targeting a different HER2 epitopes could avoid these side effects. Accordingly, there remains an urgent need for agents targeting HER2. [0010] The EGFR family of receptor tyrosine kinases are important regulators of cell growth and proliferation. One member of the family, ErbB2, has been implicated in a host of disorders and diseases including many forms of cancer.
[0011] Accordingly, there is an urgent need for therapeutic and diagnostic agents for detecting and treating ErbB2-mediated disorders including proliferative disorders.
Summary of the Invention
[0012] The invention relates to novel ErbB2 binding proteins that bind the extracellular domain (ECD) of ErbB2, in particular, human ErbB2. The novel binding protein can be antibody, an antigen-binding fragment of an antibody or a small modular immunopharmaceutical (SMIP). In various embodiments, the binding proteins: bind the ECD in the L1 , CR1 , L2 or CR2 domain, in some cases in the membrane proximal region of the CR2 domain, such as a membrane proximal region comprising the amino acid sequence shown in the first 12 residues of SEQ ID NO: 671 (i.e., without the EKK). In some embodiments, a HER2 binding protein of the invention is an ErbB2 agonist, increases tyrosine phosphorylation of ErbB2 and/or of AKT, MAP kinase (MAPK), MEK kinase, ERK 1/2, preferentially binds ErbB2 ECD homodimer over monomer or shed ECD, binds HER2 on cells and in some cases internalizes, decreases shedding of ErbB2 ectodomain shedding compared to shedding from cells of the same type without a bound HER2 binding protein of the invention, reduces the amount of cell surface HER2, reduces ErbB2 mediated proliferation of cancer cells, increases apoptosis in cancer cells, increases the number of cells in S phase after treatment with the binding protein, reduces tumor growth in vivo, enhances the effectiveness of some other anti-proliferative or cytotoxic agents or any combination of these properties.
[0013] The invention further relates to nucleic acids encoding the binding proteins or their components, vectors and host cells comprising the nucleic acids and methods of producing the binding proteins by expressing them in the host cells.
[0014] In a further aspect, the invention provides kits and compositions comprising one or more binding proteins of the invention and in some embodiments, further comprising an additional component that is a therapeutic or diagnostic agent, particularly a chemotherapeutic agent.
[0015] The invention also provides methods for producing and identifying binding proteins of the invention and methods for using them, including for treating cancer or other ErbB2 mediated disorders in a subject in need thereof, for reducing proliferation of and/or increasing apoptosis in ErbB2 expressing cells, including cancer cells, for reducing tumor growth and for diagnostic uses, including detecting and/or quantifying the presence of ErbB2 or cells expressing it.
Brief Description of the Figures
[0012] Figure 1. Schematic representation of the selection strategy used in the generation of human anti-Her2 scFv binding domains. [0013] Figure 2 (A-M). Alignments of the heavy chain amino acid sequences of human anti-Her2 scFvs with the germline human VH gene sequence. CDRs are in bold type.
[0014] Figure 3 (A-L). Alignments of the light chain amino acid sequences of human anti-Her2 scFvs with the germline human Vκ or Vλ sequence. CDRs are in bold type.
[0015] Figure 4. (A) Schematic diagram of the protein constructs used for selection and screening of scFvs and SMIPs that bind to the extracellular domain of Her2. (B) scFvs and SMIPs are binned into 4 distinct groups according to their binding phenotype as determined using the reagents in Fig 4A. (* Herceptin contact sites)
[0016] Figure 5. ELISA data for scFv binding to Her2. Binding data for phage- expressed scFv binding to Her2-expressing cells is shown on the left side of the table and data for soluble scFv binding to purified Her2 proteins is shown on the right. ELISA data is scored using a range that correlates with binding signal as indicated by -, + etc.
[0017] Figure 6. Binding of HER2 SMIPs (HER067 and HER030), HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) to (A) HER2 dimer; (B) HER2 monomer; and (C) HER2 shed ectodomain found in SKBR3 supernatant.
[0018] Figure 7. ELISA and BIACORE® data for HERCEPTIN® (trastuzumab) and SMIPs binding to Her2. Graphs represent binding of HERCEPTIN® (trastuzumab), HerO33 or Her030 binding to various Her2 proteins determined by standard ELISA methods. The table represents Kd values for HERCEPTIN® (trastuzumab), HerO33, Her030 and HerO18 (Herceptin SMIP) binding to various Her2 proteins as detected by BIACORE®.
[0019] Figure 8 provides a summary of various specific SMIPs, HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) binding to various HER2 molecules (different sizes and different species, including human, murine, and macaque) as well as binding to several different cancer cell lines. [0020] Figures 9A-9H show cell surface binding of HER2 SMIPs (HER067 and
HER094), HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) to cell lines (A) Ramos (Her27CD20+ control); (B) BT474; (C) 22rv1 ; (D) MDA-MB-175; (E) MDA-MB-361 (ATCC); (F) MDA-MB-453; (G) MDA-MB-361 (JL); and (H) SKBR3.
[0021] Figure 10 provides a summary of the anti-proliferative activity of HER033 SMIP and HERCEPTIN ® (trastuzumab) on several different cancer cell lines.
[0022] Figure 11. Proliferation of MDA-MB-361 cells following treatment with HER030 or HER033. MDA-MB-361 (ATCC) breast cancer cells were plated in 96-well format and treated with 0-10 ug/ml anti-Her2 or control reagents for 72 hr. Cells were washed, fixed, and stained with DAPI. Stained nuclei were counted using Cellomics High Content assay measuring fluorescence at 36OnM.
[0023] Figure 12 provides a summary of the anti-proliferative activity of various specific SMIPs, HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) on several different cancer cell lines.
[0024] Figure 13. Western blot analysis of effect of HerO33 on Her2 receptor phosphorylation (Y1248) following 24hr treatment of MDA-MB-361 breast cancer cells. Cells were treated in vitro with HerO33, HERCEPTIN® (trastuzumab), or a small molecule Her2 kinase inhibitor for 24hrs either alone or in the presence of heregulin (HRG1 10ng/ml) activation of Her3. Protein lysates (50ug/well) were size fractionated by SDS-PAGE, transferred to nitrocellulose and probed with anti-phospho-Her2(Y1248) antibody. Inhibition of the Her2 receptor kinase blocked the endogenous Her2 autophosphorylation at tyrosine 1248 relative to control. Treatment with Herceptin did not significantly modulate receptor phosphorylation whereas treatment with HerO33 stimulated Her2 receptor phosphorylation. Western blots were subsequently reprobed with anti-Actin antibody as protein loading control. [0025] Figure 14. HerO33 increases downstream phosphoprotein signal transduction in MDA-MB-361 and BT474 breast cancer cells. Cells were plated in 96-well format and treated with anti-Her2 reagents or Heregulin for 10 minutes. Cells were stained with either rabbit anti-pAKT, anti-pERK, anti-pS6K, or anti-p38MAPK antibodies and ALEXA594 labeled secondary antibody and cellular fluorescence quantified by high content (Cellomics) analysis. In both breast cancer cell lines, treatment with HerO33 SMIP induces phosphorylation of AKT and ERK proteins similar to treatment with the Her3 ligand Heregulin. MDA-MB-361 cells also demonstrate significant activation of p38MAP kinase.
[0026] Figure 15. Kinetic analysis of HerO33 stimulated downstream effector phosphorylation in MDA-MB-361 breast cancer cells. Cells were grown in 96-well format and treated with either anti-Her2 reagents or Her3 ligand Heregulin for 10min to 24hr as indicated. Cells were stained with either rabbit anti-pAKT, anti-pERK, anti-pS6K, or anti- p38MAPK antibodies and ALEXA594 labeled secondary antibody and cellular fluorescence quantified by high content (Cellomics) analysis. HerO33 treatment induces sustained activation of AKT, ERK and p38MAP kinase phosphorylation in this cell line similar in magnitude to levels following stimulation with 10ng/ml Heregulin.
[0027] Figures 16A and 16B show level of phosphorylation of ErbB2, and ERK1/2 in MDA-MB-361 cells when treated with HER2 SMIP HER067, HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018).
[0028] Figure 17 shows the effect on cell cycle of HER033 SMIP, HERCEPTIN® (trastuzumab), and heregulin on the SKBR3 and BT474 cell lines.
[0029] Figure 18 shows the effect on cell cycle of HER033 SMIP, HERCEPTIN® (trastuzumab), and heregulin on the MDA-MB-453 and MDA-MB-361 cell lines.
[0030] Figure 19. MDA-MB-361 xenograft progression in irradiated nu/nu mice. Female nu/nu mice were exposed to 400 rads of total body irradiation. After three days, they were injected subcutaneously in the dorsal right flank with 1x107 MDA-MB-361 cells in Matrigel. When the tumors had reached a mass of 0.1-0.25 g, animals were dosed with Herceptin, HER033, or vehicle (100 ug/mouse, intraperitoneally) on days 1 , 4, 6, 8 and 11 (n=10 mice/treatment group). Tumors were measured, and calculated tumor volumes for individual mice are shown for animals treated with vehicle (A), Herceptin (B), or HER033 (C). Animals developing tumors larger than 2.5 g were sacrificed. The mean tumor volume +SEM are plotted in (D). Means were not calculated for treatment groups in which animals with large tumors had been sacrificed.
[0031] Figure 20. MDA-MB-361 xenograft progression in Balb/c nude mice. Male
Balb/c nude mice were injected subcutaneously in the dorsal right flank with 1x107 MDA-MB- 361 cells in Matrigel. When the tumors had reached a mass of 0.1-0.25 g, animals were dosed with HERCEPTIN® (trastuzumab), HER033, or vehicle (100 ug/mouse, intraperitoneally) on days 1 , 4, 6, 8 and 11 (n=10 mice/treatment group). Tumors were measured, and calculated tumor volumes for individual mice are shown for animals treated with vehicle (A), HERCEPTIN® (trastuzumab) (B)1 or HER033 (C). Animals developing tumors larger than 2.5 g were sacrificed. The mean tumor volume +SEM are plotted in (D).
Means were not calculated for treatment groups in which animals with large tumors had been sacrificed.
[0032] Figures 21 and 22 show the in vivo efficacy of HER2 SMIP
HER033/HER067 when used to treat SCID-Beige having a tumor xenograft of MDA-MB-361 cells and the in vitro anti-proliferative activity on MDA-MB-361 cells. The top panel of Figure
21 shows the mean tumor volume in mice treated with HER033 SMIP1 HERCEPTIN®
(trastuzumab), or vehicle (IgG) after 21 days. The bottom panel of Figure 21 shows a titration of anti-proliferative activity of HER2 SMIPs (HER067 and HER094) and trastuzumab
SMIP (HER018) on the MDA-MB-361 cells used for xenografting in the mice. Figure 22 shows the tumor volume of individual mice in each treatment group.
[0033] Figure 23 (A-M). Alignments of the heavy chain amino acid sequences of human anti-ERBB2 antibodies with the germline human VH gene sequence. CDRs are in bold type.
[0034] Figure 24 (A-M). Alignments of the light chain amino acid sequences of human anti-ERBB2 antibodies with the germline human Vx or Vλ sequence. CDRs are in bold type.
[0035] Figures 25A and 25B. Figure 25A is a schematic representation of the
"stumpy" strategy used in the generation of human anti-ERBB2 antibodies. Figure 25B shows the predicted structure of the "stumpy peptide" used for selection. The EKK sequence at C terminus maintains the helical structure predicted from the NMR (Goetz et al.,
2001. Biochemistry 40: 6534-6540).
[0036] Figure 26 (A-K). Alignments of the heavy chain and light chain amino acid sequences of human anti-ERBB2 antibodies with the germline human VH gene sequence.
CDRs are in bold type. The human anti-ERBB2 antibodies were selected using the "stumpy" strategy. [0037] Figure 27 shows various HER2 soluble protein constructs used to investigate binding of molecules of the invention.
[0038] Figure 28 provides a summary of various specific SMIPs1 HERCEPTIN® (trastuzumab), and a trastuzumab SMIP (HER018) binding to various HER2 molecules (different sizes and different species, including human, murine, and macaque) as well as binding to Her2 monomers and shed extracellular domain.
[0039] Figure 29 is a graphical representation of different SMIPs binding to various Her2 molecules.
[0040] Figure 30 graphically depicts the binding of anti-HER2 "stumpy" binders (HER085, HER156 and HER 169) to soluble HER2 constructs.
[0041] Figure 31 summarizes the cell surface binding of various HER2 SMIPs to different cell lines.
[0042] Figure 32 is a bar graph showing cell staining of JIMT-1 cells with severalanti-HER2 SMIPS including "stumpy" binders. [0043] Figure 33 graphically depicts staining of various cell lines with HER146,
HER156 and HER169.
[0044] Figure 34 summarizes the cross-reactivity of various HER2 SMIPs to Macaca Her2 and Murine Her2.
[0045] Figure 35 shows BIACORE® data for HERCEPTIN® (trastuzumab) and SMIPs binding to soluble Her2 proteins.
[0046] Figure 36 shows a titration of anti-proliferative activity of HER2 SMIPs (Her147, HeM 02, HeM 24, HerO67, HeM 46, HeM 16, HerO94, and HeM 33), trastuzumab SMIP (HER018) and Herceptin on MDAMB361 (ATCC) cells.
[0047] Figure 37 shows a titration of anti-proliferative activity of HER2 SMIPs (HeM 46, HerO67, HerO94, and HeM 16), trastuzumab SMIP (HER018) and Herceptin on MDAMB361 (JL) cells.
[0048] Figure 38 is a graph showing decreased proliferation of :MDA_MB-361 cells by anti-HER2 SMIPS HER146 and HER116.
[0049] Figure 39 is a table summarizing the anti-proliferative activity of various specific SMIPs, HERCEPTIN® (trastuzumab), and trastuzumab SMIP (HER018) on several different cancer cell lines.
[0050] Figure 40 is a graph showing the effect of MEK kinase inhibitor (CL-1040) on anti-HER2 SMIP anti-proliferative activity in MDA-MB-361 ATCC breast cancer cells. [0051] Figure 41 is a graph showing the effect of ERK1/2 kinase inhibitor (FR180204) on anti-HER2 SMIP anti-proliferative activity in MDA-MB-361 ATCC breast cancer cells.
[0052] Figure 42 is a graph showing the effect of ERK1 or ERK2 knockdown by RNA interference on anti-HER2 SMIP anti-proliferative activity in MDA-MB-361 ATCC breast cancer cells.
[0053] Figure 43 is an image of a Western blot showing the presence of phosphorylated HER2 at 24 hrs and 48 hrs after treatment of MDA-MB-361 ATCC breast cancer cells with HER033 SMIP or HER146 SMIP. [0054] Figures 44A and 44B show the effect on cell cycle of various SMIPs on the
(A) SKBR3 (24 hours) and (B) BT474 (24 hours) cell lines. Samples in bold are statistically higher than the controls. Samples followed by "**" are statistically lower than the controls (student T test with an error rate of 0.05).
[0055] Figures 45A-E show the effect on cell cycle of various SMIPs (A) MDA-MB- 453 (24 hours), (B) MDA-MB-361 (JL) (24 hours), (C) MDA-MB-361 (JL) (48 hours), (D) MDA-MB-361 (ATCC) (24 hours), (E), and MDA-MB-361 (ATCC) (48 hours). Samples in bold are statistically higher than the controls. Samples followed by "**" are statistically lower than the controls (student T test with an error rate of 0.05).
[0056] Figure 46 is a graph of the mean tumor volume over time after treatement in vivo with anti-HER2 SMIPs HER146 and HER116 in SCID-Beige mice having an MDA- MB-361 (JL) cells tumor xenograft. HERCEPTIN® (trastuzumab) and vehicle (IgG) are positive and negative controls, respectively!
[0057] Figure 47 presents results in SCID-Beige mice having a tumor xenograft of MDA-MB-361 (JL) cells following treatment with HER146 SMIP and HER116 SMIP. The left panel shows the survival of mice treated with HER146 SMIP, HER116 SMIP, HERCEPTIN® (trastuzumab), or vehicle (IgG) over a timecourse of 60 days. The right panel shows tumor free progression of mice treated with HER146 SMIP, HER116 SMIP, HERCEPTIN® (trastuzumab), or vehicle (IgG) over a timecourse of 60 days. The chart at the bottom demonstrates the mean survival time of mice used in the study. [0058] Figures 48A-D are a set of graphs of MDA-MB-361 xenograft tumor size in
Balb/C nude mice after treatment with anti-HER2 SMIP HER146. HERCEPTIN® (trastuzumab) and vehicle (IgG) are positive and negative controls, respectively.(A) summary of data from 10 mice in each treatment group; (B) data for individual mice in vehicle (negative control) group; (C) data for individual mice in HER146 treatment group; (D) data for individual mice in HERCEPTIN® (positive control) group. [0059] Figures 49A-D are a set of graphs of MDA-MB-361 xenograft tumor size in irradiated nu/nu mice after treatment with anti-HER2 SMIP HER146. HERCEPTIN® (trastuzumab) and vehicle (IgG) are positive and negative controls, respectively. (A) summary of data from 10 mice in each treatment group; (B) data for individual mice in vehicle (negative control) group; (C) data for individual mice in HER146 treatment group; (D) data for individual mice in HERCEPTIN® (positive control) group.
[0060] Figure 50 presents data from two independent experiments investigating the effect of anti-HER2 SMIPS of the invention on the shedding of HER2 ectodomain and on HER2 cell surface expression. (A) and (B) present the relative effect of various anti-HER2 SMIPS on ECD shedding as detected by ELISA. Panels (C) and (D) presents the relative effect of various anti-HER2 SMIPS on HER2 expression.
[0061] Figure 51 presents data from the anti-HER2 SMIP cross-blocking experiments.(A) HERCEPTIN®; (B) HER018; (C) HER067; (D) HER094; (E) HER102; (F) HER116; (G) HER146; (H) RITUXAN® and anti-CD20 SMIP (negative control). [0062] Figure 52 is a chart summarizing the cross-blocking results.
[0063] Figures 53 provide photographs depicting the internalization of anti-HER2 SMIP (panels A and B) and cell surface HER2 (panel C).
[0064] Figure 54 is a graph depicting Fc dependent cellular cytoxicity (FcDCC) of various anti-HER2 SMIPS in MDA-MB-361 -JL and SKBR3 cells. [0065] Figure 55 is a graph depicting complement-dependent cytotoxicity (CDC)
(complement-dependent cytotoxicity) in SKBR3 cells.
[0066] Figure 56 presents data from ELISA testing of SMIP binding to Her2-SIIS after storage of the SMIP in plasma at various temperatures and durations. (A) HerO67; (B) HeM 46. [0067] Figures 57 depict different possible ratios of SM IP/receptor complexes with their predicted mass.
[0068] Figure 58 shows the masses of SMIP/receptor complexes observed following SEC-RI-MALLS analysis.
£0069] Figures 59A-D provide a series of dose response curves of different cells pre-treated with 5-fold dilution series of HER146 and then treated with corresponding 5-fold dilution series of different chemotherapeutic agents, or combinations thereof, and charts of the dilution series times of incubation used. (A) MDA-MB-453 cells with HER146 and Cisplatin or Taxol; (B) MDA-MB-453 cells with HER146 and Doxorubicin; (C) MDA-MB-361- JL cells with Cisplatin or Taxol; (D) MDA-MB-361 -JLcells with HER146 and Doxorubicin or Gemcitabine. [0070] Figure 60 is an immunoblot with short (left) or long (right) exposures showing Her2 immunoprecipitated from Ramos or SKBR3 cell lysates by Herceptin, 3B5, HER156, or HER169.
[0071] Figure 61 is two immunoblots in color and a black-and-white exposure of the color blot on the right, showing Her2 immunoprecipitated from Ramos, JIMT-1 , or MDA- MB-361 ATCC cell lysates by human IgG1 3B5, HER116, HER156, or HER169.
Detailed Description of the Invention I. Definitions
[0072] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. The present invention provides novel binding proteins that, specifically bind the extra cellular domain (ECD) of ErbB2, especially human ErbB2. lnsome embodiments, the binding protein is an antibody or an antigen binding fragment of such antibody that specifically binds the ECD. In other embodiments, the binding protein is a small modular immunopharmaceutical (SMIP).
[0073] The term "antibody" refers to an intact four-chain molecule having 2 heavy chains and 2 light chains, each heavy chain and light chain having a variable domain and a constant domain, or an antigen-binding fragment thereof, and encompasses any antigen- binding domain. In various embodiments, an antibody of the invention may be polyclonal, monoclonal, monospecific, polyspecific, bi-specific, humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted (including CDR grafted), or an in vitro generated antibody.
[0074] The term "antigen-binding fragment" of an antibody that specifically binds the ECD of ErbB2 refers to a portion or portions of the antibody that specifically binds to the ECD. An antigen-binding fragment may comprise all or a portion of an antibody light chain variable region (VL) and/or all or a portion of an antibody heavy chain variable region (VH) so long as the portion or portions are antigen-binding. However, it does not have to comprise both. Fd fragments, for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include (1 ) a Fab fragment, a monovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) a Fd fragment having the two VH and CH1 domains; (4) a Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), that has a VH domain; (6) an isolated complementarity determining region (CDR)1 and (7) a single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.
[0075] The term "effective amount" refers to a dosage or amount that is sufficient to alter ErbB2 activity, to ameliorate clinical symptoms or achieve a desired biological outcome, e.g., decreased cell growth or proliferation, decreased heterodimerization with another member of the EGF family decreased homodimerization, decrease tumor growth rate or tumor size, increased cell death etc.
[0076] The term "human antibody" includes antibodies having variable and constant region sequences corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (See Kabat, et al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The amino acid sequences of a human antibody, when aligned with germline immunoglobulin sequences, most closely align with human immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Such non-germline residues may occur in a framework region, a CDR, for example in the CDR3, or in the constant region. A human antibody can have one or more residues, such as any number from 1-15, including all of the integers between 1 and 15, or more, replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence. CDRs are as defined by Kabat or in Chothia C, Lesk AM, Canonical structures for the hypervariable regions of immunoglobulins, J MoI Biol. 1987 Aug 20;196(4):901-17. [0077] The phrase "inhibit" or "antagonize" an ErbB2/HER2 activity refers to a reduction, inhibition, or otherwise diminution of at least one activity of ErbB2 due to binding an anti-ErbB2 antibody or antigen binding portion, wherein the reduction is relative to the activity of ErbB2 in the absence of the same antibody or antigen-binding portion. The activity can be measured using any technique known in the art, including, for example, as described in the Examples. Activation of the Her2 receptor tyrosine kinase can be measured by the degree of phosphorylation of key tyrosine residues in the intracellular domain. For example, Tyr1248 is a known site of autophosphorylation and thus is a direct measure of Her2 receptor kinase activity. Typically the degree of phosphorylation can be determined by Western blot analysis probing with anti-phopho-Her2 specific antibodies (eg. Tyr1248, Tyr1139, Tyr1112, Tyr877, Tyr1221/1222). Alternatively, cells can be permeabilized and probed with fluorescently labeled phospho-Her2 antibodies and measured either by flow cytometry or high content (Cellomics) analysis. Additionally, the Her2 receptor can be immunoprecipitated, digested with trypsin protease and the degree of phosphorylation at specific sites within the individual Her2 peptides determined by standard Mass Spec techniques. Inhibition or antagonism does not necessarily indicate a total elimination of the ErbB2 polypeptide biological activity. In some embodiments, the reduction in activity may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, including 100% reduction, i.e., elimination of the activity.
[0078] The term "ErbB2" refers to erythroblastic leukemia viral oncogene homolog 2. In the case of human ErbB2, it also is known as c-erb-B2 or HER2/neu. In some embodiments the ErbB2 may comprise: (1 ) an amino acid sequence of a naturally occurring mammalian ErbB2 polypeptide (full length or mature form) or a fragment thereof, or a fragment thereof; (2) an amino acid sequence substantially identical to, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to said amino acid sequence or a fragment thereof; (3) an amino acid sequence that is encoded by a naturally occurring mammalian ErbB2 nucleotide sequence or a fragment thereof, or (4) a nucleotide sequence that hybridizes to the foregoing nucleotide sequence under stringent conditions, e.g., highly stringent conditions.
[0079] HER2 or c-erb-B2 encodes a transmembrane receptor protein of 185 kDa, which is structurally related to the epidermal growth factor receptori . HER2 protein overexpression is observed in 25%-30% of primary breast cancers and is associated with decreased overall survival and a lowered response to chemotherapy and hormonal therapy, which can continue throughout the course of the disease and drives aggressive tumor growth. [0080] The term "ErbB2 activity" refers to at least one cellular process initiated or interrupted as a result of ErbB2 binding to a receptor complex comprising ErbB2 and an ErbB receptor family member including ErbB1 (EGFR), ErbB2, ErbB3, ErbB4 or comprising an ErbB ligand such as but not limited to EGF, TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 on the cell. ErbB2 activity can be determined using any suitable assay methods, for example, protein overexpression can be determined using immunohistochemistry (IHC) and may also be inferred when HER2 gene amplification is identified using fluorescence in situ hybridization (FISH).
[0081] As used herein, "in vitro generated antibody" refers to an antibody where all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection (e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen). This term excludes sequences generated by genomic rearrangement in an immune cell.
[0082] The term "isolated" refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived. The term also refers to preparations where the isolated protein is sufficiently pure for pharmaceutical compositions; or at least 70-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
[0083] The phrase "percent identical" or "percent identity" refers to the similarity between at least two different sequences. This percent identity can be determined by standard alignment algorithms, for example, the Basic Local Alignment Tool (BLAST) described by Altshul et al. ((1990) J. MoI. Biol., 215: 403-410); the algorithm of Needleman et al. ((1970) J. MoI. Biol., 48: 444-453); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4: 11-17). A set of parameters may be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) that has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity is usually calculated by comparing sequences of similar length.
[0084] The terms "specific binding" or "specifically binds" refer to forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the association constant KA is higher than 106 M"1. The appropriate binding conditions, such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques. An antibody is said to specifically bind an antigen when the K0 is ≤ 1 mM, preferably ≤ 10O nM. [0085] As used herein, the term "stringent" describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. One example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2X SSC, 0.1% SDS at 500C. A second example of stringent hybridization conditions is hybridization in 6X SSC at about 45°C, followed by at least one wash in 0.2X SSC, 0.1% SDS at 55°C. Another example of stringent hybridization conditions is hybridization in 6X SSC at about 45°C, followed by at least one wash in 0.2X SSC, 0.1 % SDS at 6O0C. A further example of stringent hybridization conditions is hybridization in 6X SSC at about 450C, followed by at least one wash in 0.2X SSC, 0.1% SDS at 65°C. High stringent conditions include hybridization in 0.5M sodium phosphate, 7% SDS at 65°C, followed by at least one wash at 0.2X SSC1 1 % SDS at 65°C. [0086] The phrase "substantially as set out," "substantially identical" or
"substantially homologous" means that the relevant amino acid or nucleotide sequence (e.g., CDR(s), VH, or VL domain) will be identical to or have insubstantial differences (through conserved amino acid substitutions) in comparison to the sequences that are set out. Insubstantial differences include minor amino acid changes, such as 1 or 2 substitutions in a 5 amino acid sequence of a specified region. In the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity of the first antibody.
[0087] Sequences substantially identical or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiment, the sequence identity can be about 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity or homology exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
[0088] The term "therapeutic agent" is a substance that treats or assists in treating a medical disorder. Therapeutic agents may include, but are not limited to, anti-proliferative agents, anti-cancer agents including chemotherapeutics, anti-virals, anti-infectives, immune modulators, and the like that modulate immune cells or immune responses in a manner that complements the ErbB2 activity of an anti-ErbB2 binding protein of the invention. Non- limiting examples and uses of therapeutic agents are described herein. [0089] As used herein, a "therapeutically effective amount" of an anti-ErbB2 binding protein refers to an amount of an binding protein that is effective, upon single or multiple dose administration to a subject (such as a human patient) at treating, preventing, curing, delaying, reducing the severity of, and/or ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.
[0090] The term "treatment" refers to a therapeutic or preventative measure. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay, reduce the severity of, and/or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
II. Anti-ErbB2 Binding Proteins
[0091] In a first aspect, the invention provides novel ErbB2/HER2, particularly human ErbB2/HER2, ErbB2/HER2 binding proteins that bind in the extra-cellular domain
(ECD). In various embodiments, the binding proteins of the invention bind in the LR1 , CR1 , LR2 or CR2 domain of the ECD, including a membrane proximal region of CR2 comprising the amino acid sequence in the first twelve residues of SEQ ID NO: 671 (i.e., without the EKK). Unlike HERCEPTIN®, in some embodiments the binding proteins of the invention preferentially bind ErbB2 nomodimers over monomers or shed ECD. In some embodiments, the binding proteins of the invention bind ECD homodimers substantially more than monomers. In some cases, the binding protein has no appreciable or significant binding to ECD monomers or to shed ECD.
[0092] In some embodiments, the novel binding proteins are ErbB2 agonists and increase tyrosine phosphorylation of ErbB2 and at the same time, have anti-proliferative activity and pro-apoptotic activity. In some embodiments, the binding proteinincreases kinase activity in a HER-2 expressing cell, including but not limited to increasing kinase activity of MEK, MAPK, ERK1 , ERK2 or a combination thereof.
[0093] The anti-ErbB2/HER2 binding proteins of the invention can be obtained by any of numerous methods known to those skilled in the art. For example, antibodies can be produced using recombinant DNA methods (U.S. Patent 4,816,567). Monoclonal antibodies may be produced by generation of hybridomas (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499) in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof. [0094] One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315- 1317; Clackson et al. (1991 ) Nature, 352: 624-628; Marks et al. (1991) J. MoI. Biol., 222: 581-597WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.
[0095] In addition to the use of display libraries, the specified antigen can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21 , US 2003-0070185, WO 96/34096, published Oct. 31 , 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996. [0096] The subunit structures, e.g., a CH, VH, CL, VL, CDR, FR, and three- dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. One of skill in the art will recognize that a complete 4-chain immunoglobulin comprises active portions, e.g., a portion of the VH or VL domain or a CDR that binds to the antigen, i.e., an antigen-binding fragment, or, e.g., the portion of the CH subunit that binds to and/or activates, e.g., an Fc receptor and/or complement. CDRs typically refer to regions that are hypervariable in sequence and/or form structurally defined loops, for example, Kabat CDRs are based on sequence variability, as described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al, or alternatively, to the location of the hypervariable structural loops as described by Chothia. See, e.g., Chothia, D. et al. (1992J J. MoI. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Still another standard is the AbM definition used by Oxford Molecular's AbM antibody modelling software, which defines the contact hypervariable regions based on crystal structure. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Embodiments described with respect to Kabat CDRs can alternatively be implemented using similar described relationships with respect to Chothia hypervariable loops or to the AbM- defined loops. [0097] In another embodiment, a monoclonal antibody is obtained from the non- human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81 :6851 , 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B. Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen. [0098] Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques ΛWA; and by US 5,585,089; US 5,693,761 ; US 5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.
[0099] In certain embodiments, a humanized antibody is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations. Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymoi, 92: 3-16, 1982), and may be made according to the teachings of PCT Publication WO92/06193 or EP 0239400).
[0100] An antibody or fragment thereof may also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class Il binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class Il DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. Human germline sequences, e.g., are disclosed in Tomlinson, et al. (1992) J. MoI. Biol. 227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al. (1992) J. MoI. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, I.A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, e.g., as described in U.S. Patent No. 6,300,064. [0101] In certain embodiments, an antibody can contain an altered immunoglobulin constant or Fc region. For example, an antibody produced in accordance with the teachings herein may bind more strongly or with more specificity to effector molecules such as complement and/or Fc receptors, which can control several immune functions of the antibody such as effector cell activity, lysis, complement-mediated activity, antibody clearance, and antibody half-life. Typical Fc receptors that bind to an Fc region of an antibody (e.g., an IgG antibody) include, but are not limited to, receptors of the FcγRI, FcγRII, and FcγRI 11 and FcRn subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc receptors are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92, 1991 ; Capel et al., lmmunomethods 4:25-34,1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41 , 1995). [0102] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present invention is not necessarily limited to any particular source, method of production, or other special characteristics of an antibody. [0103] In some embodiments, an anti-ErbB2 antibody of the invention may be a
VHH molecule. VHH molecules (or nanobodies), as known to the skilled artisan, are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains, such as those derived from Camelidae as described in WO9404678, incorporated herein by reference. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco and is sometomes called a camelid or camelized variable domain. See e.g., Muyldermans., J. Biotechnology (2001 ) 74(4):277-302, incorporated herein by reference. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain. VHH molecules are about 10 times smaller than IgG molecules. They are single polypeptides in which the CDR3 is longer than a conventional antibody, the VH:VL interface residues are different, and extra cysteines are generally present. These molecules tend to be very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHS produces high yield, properly folded functional VHHS. In addition, antibodies generated in Camelids will recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids (see WO 9749805, that is incorporated herein by reference). In additional embodiments, an anti-ErbB2 antibodies or binding fragments of the invention may include single domain antibodies such as immunoglobulin new antigen receptors (IgNARs), which are a unique group of antibody isotypes found in the serum of sharks
(Greenberg et al., Nature 374: 168-173 (1995); Nuttall et al., MoI. Immunol., 38: 313-326. (2001 )). These are bivalent molecules, targeting antigen through a single immunoglobulin variable domain (-13 kDa) displaying two complementarity determining region (CDR) loops (Roux et al., Proc. Natl. Acad. ScL, 95: 11804-11809 (1998)) and having unusually long and structurally complex CDR3s, which display a high degree of variability (Greenberg et al., 1995).
[0104] Antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D1 E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgGI , lgG2, lgG3, lgG4, IgAI , and lgA2. Each light chain includes an N terminal variable (V) domain (VL) and a constant (C) domain (Q_). Each heavy chain includes an N terminal V domain (VH), three or four C domains (CHs), and a hinge region collectively referred to as the constant region of the heavy chain. The CH domain most proximal to VH is designated as CH1. The VH and VL domains consist of four regions of relatively conserved sequences called framework regions (FR1 , FR2, FR3, and FR4), that form a scaffold for three regions of hypervariable sequences also referred to as complementarity determining regions CDRs. CDRs are referred to as CDR1 , CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain may be referred to as HCDR1 , HCDR2, and HCDR3, while CDR constituents on the light chain are referred to as LCDR1 , LCDR2, and LCDR3. CDR3 is typically the greatest source of molecular diversity within the antibody-binding site. [0105] The anti-ErbB2 binding proteins of the invention include complete 4-chain antibodies and antigen-binding fragments of complete antibodies. An antigen-binding fragment (also referred to as an antigen-binding portion) includes but is not limited to Fab, Fv and ScFv molecules. The Fab fragment (Fragment antigen-binding) consists of VH-CH1 and VL-CL domains covalently linked by a disulfide bond between the constant regions. The Fv fragment is smaller and consists of VH and VL domains non-covalently linked. To overcome the tendency of non-covalently linked domains to dissociate, a single chain Fv fragment (scFv) can be constructed. The scFv contains a flexible polypeptide that links (1) the C-terminus of VH to the N-terminus of VL, or (2) the C-terminus of VL to the N-terminus of VH. Repeating units of (Gly4Ser)_often 3 or 4 repeats may be used as a linker, but other linkers are known in the art.
[0106] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab1 fragments. See, e.g., Songsivilai & Lachmann, CHn. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). In one embodiment, the bispecific antibody comprises a first binding domain polypeptide, such as a Fab' fragment, linked via an immunoglobulin constant region to a second binding domain polypeptide.
[0107] In some embodiments, an anti-ErbB2 binding protein of the invention is a Small Modular jmmunoPharmaceuticals (SMIP™). SMIPs and their uses and applications are disclosed in, e.g., U.S. Published Patent Application. Nos. 2003/0118592, 2003/0133939, 2004/0058445, 2005/0136049, 2005/0175614, 2005/0180970, 2005/0186216, 2005/0202012, 2005/0202023, 2005/0202028, 2005/0202534, and 2005/0238646, and related patent family members thereof, all of which are hereby incorporated by reference herein in their entireties. [0108] A SMIP™ typically refers to a binding domain-immunoglobulin fusion protein that includes a binding domain polypeptide that is fused or otherwise connected to an immunoglobulin hinge or hinge-acting region polypeptide, which in turn is fused or otherwise connected to a region comprising one or more native or engineered constant regions from an immunoglobulin heavy chain, other than CH1 , for example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE (see e.g., U.S. 2005/0136049 by Ledbetter, J. et a/., which is incorporated by reference, for a more complete description). The binding domain-immunoglobulin fusion protein can further include a region that includes a native or engineered immunoglobulin heavy chain CH2 constant region polypeptide (or CH3 in the case of a construct derived in whole or in part from IgE) that is fused or otherwise connected to the hinge region polypeptide and a native or engineered immunoglobulin heavy chain CH3 constant region polypeptide (or CH4 in the case of a construct derived in whole or in part from IgE) that is fused or otherwise connected to the CH2 constant region polypeptide (or CH3 in the case of a construct derived in whole or in part from IgE). Typically, such binding domain-immunoglobulin fusion proteins are capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytotoxicity, complement fixation, and/or binding to a target, for example, a target antigen, such as human ErbB2.
[0109] The binding domain of a SMIP of the invention may contain a complete VH and a complete VL joined by linker antigen-binding portions of a VH and/or VL and may V2 or be linked in either orientation, i.e., VH-linker-VL or VL-linker-VH. Any suitable linker can be used in a SMIP of the invention and will be known to those of skill in the art. Exemplary linkers may be found, for example in WO 2007/146968 Tables 5 and 10-12 of which are incorporated by reference in their entirety. Likewise, any immunoglobulin hinge sequence or hinge-acting sequence may be used in a SMIP of the invention. [0110] In some SMIP embodiments at least one of the immunoglobulin heavy chain constant region polypeptides (i.e., CH2, CH3 or CH4) is from a human immunoglobulin heavy chain. In various embodiments, the immunoglobulin heavy chain constant region polypeptides are of an isotype selected from human IgG and human IgA. In certain further embodiments of the above described SMIP1 the linker polypeptide comprises at least one polypeptide having as an amino acid sequence (GIy4, Ser) and in certain other embodiments the linker polypeptide comprises at least three repeats of said polypeptide. In certain embodiments the immunoglobulin hinge region polypeptide comprises a human IgA hinge region polypeptide.
[0111] An immunoglobulin hinge region polypeptide, as discussed above, includes any hinge peptide or polypeptide that occurs naturally, as an artificial peptide or as the result of genetic engineering and that is situated in an immunoglobulin heavy chain polypeptide between the amino acid residues responsible for forming intrachain immunoglobulin-domain disulfide bonds in CH 1 and CH2 regions; hinge region polypeptides for use in the present invention may also include a mutated hinge region polypeptide. Accordingly, an immunoglobulin hinge region polypeptide may be derived from, or may be a portion or fragment of (i.e., one or more amino acids in peptide linkage, typically 5-65 amino acids, preferably 10-50, more preferably 15-35, still more preferably 18-32, still more preferably 20- 30, still more preferably 21 , 22, 23, 24, 25, 26, 27, 28 or 29 amino acids) an immunoglobulin polypeptide chain region classically regarded as having hinge function, as described above. But, a hinge region polypeptide for use in the instant invention need not be so restricted and may include amino acids situated (according to structural criteria for assigning a particular residue to a particular domain that may vary, as known in the art) in an adjoining immunoglobulin domain such as a CH1 domain or a CH2 domain, or in the case of certain artificially engineered immunoglobulin constructs, an immunoglobulin variable region domain.
[0112] Wild-type immunoglobulin hinge region polypeptides include any naturally occurring hinge region that is located between the constant region domains, CH 1 and CH2, of an immunoglobulin. The wild-type immunoglobulin hinge region polypeptide is preferably a human immunoglobulin hinge region polypeptide, preferably comprising a hinge region from a human IgG immunoglobulin, and more preferably, a hinge region polypeptide from a human IgGI isotype. As is known to the art, despite the tremendous overall diversity in immunoglobulin amino acid sequences, immunoglobulin primary structure exhibits a high degree of sequence conservation in particular portions of immunoglobulin polypeptide chains, notably with regard to the occurrence of cysteine residues which, by virtue of their sulfyhydryl groups, offer the potential for disulfide bond formation with other available sulfydryl groups. Accordingly, in the context of the present invention wild-type immunoglobulin hinge region polypeptides may be regarded as those that feature one or more highly conserved (e.g., prevalent in a population in a statistically significant manner) cysteine residues, and in certain preferred embodiments a mutated hinge region polypeptide may be selected that contains zero or one cysteine residue and that is derived from such a wild-type hinge region.
[0113] A mutated immunoglobulin hinge region polypeptide may comprise a hinge region that has its origin in an immunoglobulin of a species, of an immunoglobulin isotype or class, or of an immunoglobulin subclass that is different from that of the CH2 and CH3 domains. For instance, in certain embodiments of the invention, the SMIP may comprise a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide comprising a wild-type human IgA hinge region polypeptide, or a mutated human IgA hinge region polypeptide that contains zero or only one cysteine residues, as described herein. Such a hinge region polypeptide may be fused to an immunoglobulin heavy chain CH2 region polypeptide from a different Ig isotype or class, for example an IgG subclass, which in certain preferred embodiments will be the IgGI subclass.
[0114] In some embodiments, an anti-ErbB2 antibody of the invention is a VHH molecule. VHH molecules (or nanobodies), as known to the skilled artisan, are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains, such as those derived from Camelidae as described in WO9404678, incorporated herein by reference. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco and is sometomes called a camelid or camelized variable domain. See e.g., Muyldermans., J. Biotechnology (2001 ) 74(4):277-302, incorporated herein by reference. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain. VHH molecules are about 10 times smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHS produces high yield, properly folded functional VHHS. In addition, antibodies generated in Camelids will recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids (see WO 9749805, that is incorporated herein by reference). [0115] Amino acid (AA) sequences of illustrative heavy chain variable domains (VH) and light chain variable domains (VL) of the anti-ErbB2 antibodies of this invention, are set forth in the attached Sequence Table. Table 1 provides the Sequence Identifiers (SEQ ID Nos) of the VH and VL domains. One hundred specific embodiments of the antibodies are identified as: S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1 R2C_CS_1 H12, S1 R2A_CS_1 D3, S1 R3B2_BMV_1 E1 , S1 R3C1_CS_1 D3, S1 R3B2_DP47_1 E8, S1 R3B2_BMV_1 G2, S1R3B2_BMV_1 H5, S1 R3C1_CS_1A6, S1 R3B2_DP47_1C9, S1 R3B2_DP47_1 E10, S1 R3C1_CS_1 B10, S1R3A1_BMV_1 F3, S1 R3B1_BMV_1G11 , S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1 R3A1_DP47_11 B7, S1 R3A1_DP47_11 D1 , S1 R3A1_DP47_7F3, S1 R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1R2A_CS_5A1, S1R2A_CS_8C8, S1R3A1_CS_13H5, S1 R2B_CS_5E9, S1 R3A1_CS_8F9, S1 R3A1_CS_14B5, S1 R2A_CS_9E10,
S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1R2A_CS_7D4, S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1E11, S5R2_DP47_1H11, S6R3_CS_1G5, S6R2_DP47_1H11, S5R3_DP47_1A10, S5R2_DP47_1D11, S5R2_CS_1 A8, S6R3_CS_1 B7, S6R2_CS_1 E5, S6R3_BMV_1 C2, S5R2_DP47_1 B101 S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1 H9.
Table 1:
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000028_0001
Figure imgf000029_0001
[0116] According to the nomenclature used herein, "S1 R2A_CS_1 F7" indicates clone 1 F7 from round 2A of the first selection from the CS library.
[0117] An anti-ErbB2 binding protein of this invention may optionally comprise antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to a light chain constant domain which can be a CK or a Cλ. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain constant region, which can be a IgA, IgD, IgE, IgG, or IgM constant region or any isotype subclass including IgGI, lgG2, lgG3, lgG4, IgAI or lgA2. Constant region sequences are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No.91-3242, National Institutes of Health Publications, Bethesda, MD (1991)). Therefore, binding proteins within the scope of this invention may include VH and VL domains, or a portion thereof, combined with constant regions or portions thereof known in the art.
[0118] In certain embodiments of the invention, the ErbB2 binding protein comprises a VH domain, a VL domain, or a combination thereof, comprising the VH or VL amino acid sequence, respectively, found in any one of S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1R2C_CS_1D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3, S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1 R3A1_DP47_7F3, S1 R2B_DP47_4E3, S1 R3C1_DP47_2G2, S1 R3A1_DP47_11 H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1 R3A1_CS_11 C3, S1 R3A1_CS_13H11 , S1 R3A1_CS_2D9, S1 R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1 R2A_CS_5A1 , S1R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9, S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1R2A_CS_7D4, S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1 E11 , S5R2_DP47_1 H11 , S6R3_CS_1 G5, S6R2_DP47_1 H11 , S5R3_DP47_1A10, S5R2_DP47_1D11, S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1H9. In some embodiments, the VH and VLare from the same reference antibody. That is, an anti-ErbB2 binding protein of the invention may comprise both the VH and VL amino acid sequence of one of the above-listed antibodies.
[0119] An anti-ErbB2 antibody of the invention may comprise one, two, three, four, five or all six complementarity determining regions (CDRs) from any one of the above-listed antibodies. In some embodiments, an anti-ErbB2 binding protein of the invention comprises the HCDR1, HCDR2 and HCDR3 (heavy chain CDR set), the LCDR1, LCDR2 and LCDR3 (light chain CDR set) or both the heavy chain CDR set and the light chain CDR set of one of the anti-ErbB2 antibodies exemplified herein. [0120] A CDR3 sequence found in any one of the specifically exemplified anti-
ErbB2 antibodies are encompassed within the scope of this invention. For example, in one embodiment, an anti-ErbB2 binding protein of the invention comprises an HCDR3 amino acid sequence found in any one of S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3, S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6,
S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1,
S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1 R3A1_DP47_11 F6, S1 R3A1_DP47_11 D3, S1 R3A1_CS_8A8, S1 R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1 R3A1_CS_15C2, S1 R3A1_CS_9C1 , S1 R2A_CS_5A1 , S1 R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9, S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1R2A_CS_7D4, S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1E11, S5R2_DP47_1H11, S6R3_CS_1G5, S6R2_DP47_1H11, S5R3_DP47_1A10,
S5R2_DP47_1D11, S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1H9.
[0121] In certain embodiments, the VH and/or VL domains may be germlined, i.e., the framework regions (FR) of these domains are mutated using conventional molecular biology techniques to match the germline sequence. In other embodiments, the FR sequences remain diverged from the consensus germline sequences.
[0122] In one embodiment, mutagenesis is used to make an antibody more similar to one or more germline sequences. This may be desirable when mutations are introduced into the framework region of an antibody through somatic mutagenesis or through error prone PCR. Germline sequences for the VH and VL domains can be identified by performing amino acid and nucleic acid sequence alignments against the VBASE database (MRC Center for Protein Engineering, UK). VBASE is a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences, including those in the current releases of the Genbank and EMBL data libraries. In some embodiments, the FR regions of the scFvs are mutated in conformity with the closest matches in the VBASE database and the CDR portions are kept intact.
[0123] In certain embodiments, an anti-ErbB2 binding of this invention specifically binds the same epitope as, competes with or cross-competes with an antibody selected from the group consisting of: S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3,
S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1 R2B_CS_6H11 , S1 R3A1_DP47_10G1 , S1 R3A1_DP47_7C1 , S1 R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1R2A_CS_5A1, S1R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9, S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1 R2A_CS_7D4, S1 R3A1_CS_15B8, S6R3_DP47_1 A10, S6R2_DP47_1 E11 , S5R2_DP47_1H11, S6R3_CS_1G5, S6R2_DP47_1H11, S5R3_DP47_1A10, S5R2_DP47_1D11, S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1 C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1 H9, for binding to ErbB2. In some embodiments, such competing or ErbB2-mediated cross- competing binding protein is an ErbB2 agonist and may further reduce proliferation of a cancer call, reduce the rate of growth of an ErbB2-expressing tumor and/or increases apoptosis in such cells and tumors. In some embodiments, such competing or cross- competing binding proteins bind ErbB2 ECD homo-dimers but do not bind ECD monomers or shed ECD. [0124] Such antibodies can be identified in a competitive binding assay. One can determine whether an antibody binds to the same epitope or cross competes for binding with a binding protein of the invention antibody by using methods known in the art. In one embodiment, one allows the binding protein of the invention to bind to ErbB2 under saturating conditions and then measures the ability of the test protein to bind to the ECD. If the test antibody is able to bind to the ECD at the same time as the reference binding protein, then the test antibody binds to a different epitope than the reference binding protein. However, if the test protein is not able to bind the to the ECD at the same time, then the test protein binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the binding protein of the invention. This experiment can be performed using ELISA1 RIA, BIACORE™, or flow cytometry. To test whether a binding protein cross-competes with another anti-ErbB2 binding protein, one may use the competition method described above in two directions, i.e. determining if the known binder blocks the test binder and vice versa. In a preferred embodiment, the experiment is performed using BIACORE™. [0125] In one embodiment, the association constant (KA) of an ErbB2 binding protein of the invention is at least 106 M"1. In another embodiment, the association constant of these antibodies for human ErbB2 is at least 109 M"1. In other embodiments, the association constant of these antibodies for human ErbB2 is at least 1010 M'1, at least 1011 M'1, or at least 1012 M'1. The binding affinity may be determined using techniques known in the art, such as ELISA, biosensor technology, such as biospecific interaction analysis, or other techniques including those described in this application.
[0126] In addition to sequence homology analyses, epitope mapping (see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996), and secondary and tertiary structure analyses can be carried out to identify specific 3D structures assumed by the presently disclosed antibodies and their complexes with antigens. Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11 :7-13) and computer modeling of virtual representations of the present antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). [0127] The invention further provides anti-ErbB2 binding proteins that comprise altered VH and/or VL sequence(s) compared to the sequences in Table 1. Such binding proteins may be produced by a skilled artisan using techniques well-known in the art. For example, amino acid substitutions, deletions, or additions can be introduced in FR and/or CDR regions. FR changes are usually designed to improve the stability and immunogenicity of the antibody, while CDR changes are typically designed to increase antibody affinity for its antigen. The changes that increase affinity may be tested by altering CDR sequence and measuring antibody affinity for its target (see Antibody Engineering, 2nd ed., Oxford University Press, ed. Borrebaeck, 1995).
[0128] Antibodies whose CDR sequences differ insubstantially from those found in any one of specifically exemplified anti-ErbB2 antibodies are encompassed within the scope of this invention. Typically, this involves substitution of an amino acid with an amino acid having similar charge, hydrophobic, or stereochemical characteristics. More drastic substitutions in FR regions, in contrast to CDR regions, may also be made as long as they do not adversely affect (e.g., reduce affinity by more than 50% as compared to unsubstituted antibody) the binding properties of the binding protein. Substitutions may also be made to germline the binding protein or stabilize the antigen binding site.
[0129] Conservative modifications will produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are made. In contrast, substantial modifications in the functional and/or chemical characteristics of the molecules may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (1 ) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (2) the charge or hydrophobicity of the molecule at the target site, or (3) the size of the molecule.
[0130] For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. (See, for example, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998, Adv. Biophys. 35:1-24).
[0131] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein. Exemplary amino acid substitutions include, but are not limited to, those set forth in Table 2.
Table 2: Amino Acid Substitutions
Figure imgf000035_0001
[0132] In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems.
[0133] In one embodiment, the method for making a variant VH domain comprises adding, deleting, or substituting at least one amino acid in the disclosed VH domains, and testing the variant VH domain for ErbB2 binding or modulation of ErbB2 activity.
[0134] An analogous method for making a variant VL domain comprises adding, deleting, or substituting at least one amino acid in the disclosed VL domains, and testing the variant VL domain for ErbB2 binding or modulation of ErbB2 activity.
[0135] A further aspect of the invention provides a method for preparing antibodies or antigen-binding fragments that specifically bind ErbB2. The method comprises:
(a) providing a starting repertoire of nucleic acids encoding a VH domain that lacks at least one CDR or contains at least one CDR to be replaced;
(b) inserting into or replacing the CDR region of the starting repertoire with at least one donor nucleic acid encoding an amino acid sequence as substantially set out herein for a VH CDR, yielding a product repertoire;
(c) expressing the nucleic acids of the product repertoire; (d) selecting a specific antigen-binding fragment that binds to ErbB2; and
(e) recovering the specific antigen-binding fragment or nucleic acid encoding it. [0136] In an analogous method, at least one VL CDR or VH CDR of the invention is combined with a repertoire of nucleic acids encoding a VL or VH domain , respectively, that lacks at least one CDR or contains at least one CDR to be replaced. The at least one VH or VL CDR may be a CDR1 , a CDR2, a CDR3, or a combination thereof, found in any of the specifically exemplified anti-ErbB2 antibodies.
[0137] In one embodiment, the variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and the at least one donor nucleic acid encodes a CDR3 amino acid sequence found in any one of SEQ ID Nos:1-95, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271, 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371 , 373, 375, 377, 379, 381 , 383, 385, 387, 389, 391 , 393, 395, 397, 399, 401 , 403, 405, 407, 409, 411 , 413, 415, 417, 419, 421 , 423, 425, 427, 429, 431 , 433, 435, 437, 439, 441 , 443, 445, 447, 449, 451 , 453, 455, 457, 459, 461 , 463, 465, 467, 469, 471 , 473, 475, 477, 479, 481 , 483, 485, 487, 489, 491 , 493, 495, 497, 499, 501 , 503, 507, 509, 511 , 513, 515, 517, 519, 521 , 523, 525, or substantially as found in such sequence.
[0138] In another embodiment, the variable domain includes a CDR1 to be replaced or lacks a CDR1 encoding region and the at least one donor nucleic acid encodes a CDR1 amino acid sequence found in any one of SEQ ID Nos: 1-95, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371 , 373, 375, 377, 379, 381 , 383, 385, 387, 389, 391 , 393, 395, 397, 399, 401, 403, 405, 407, 409, 411 , 413, 415, 417, 419, 421 , 423, 425, 427, 429, 431 , 433, 435, 437, 439, 441 , 443, 445, 447, 449, 451 , 453, 455, 457, 459, 461 , 463, 465, 467, 469, 471 , 473, 475, 477, 479, 481 , 483, 485, 487, 489, 491 , 493, 495, 497, 499, 501 , 503, 507, 509, 511, 513, 515, 517, 519, 521 , 523, 525. [0139] In another embodiment, the variable domain includes a CDR2 to be replaced or lacks a CDR2 encoding region and the at least one donor nucleic acid encodes a CDR2 amino acid sequence found in any one of SEQ ID Nos: 1-95, 251 , 253, 255, 257, 259, 261, 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321, 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 507, 509, 511, 513, 515, 517, 519, 521 , 523, 525.
[0140] In another embodiment, the variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and further comprises a CDR1 to be replaced or lacks a CDR1 encoding region, where the at least one donor nucleic acid encodes a CDR3 a CDR1 amino acid sequence, respectively, found in any one of SEQ ID Nos: 1-95, 251 , 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441 , 443, 445, 447, 449, 451 , 453, 455, 457, 459, 461 , 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525.
[0141] In another embodiment, the variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and further comprises a CDR2 to be replaced or lacks a CDR2 encoding region, where the at least one donor nucleic acid encodes a CDR3 or CDR2 amino acid sequence, respectively, found in any one of SEQ ID Nos: 1-95, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525. [0142] In another embodiment, the variable domain includes a CDR3 to be replaced or lacks a CDR3 encoding region and further comprises a CDR1 and a CDR2 to be replaced or lacks a CDR1 and a CDR2 encoding region, where the at least one donor nucleic acid encodes CDR3, CDR1 or CDR2 amino acid sequence, respectively, found in any one of SEQ ID Nos: 1-95, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371 , 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 , 393, 395, 397, 399, 401 , 403, 405, 407, 409, 411 , 413, 415, 417, 419, 421 , 423, 425, 427, 429, 431 , 433, 435, 437, 439, 441 , 443, 445, 447, 449, 451 , 453, 455, 457, 459, 461 , 463, 465, 467, 469, 471 , 473, 475, 477, 479, 481 , 483, 485, 487, 489, 491 , 493, 495, 497, 499, 501 , 503, 507, 509, 511 , 513, 515, 517, 519, 521 , 523, 525.
The present invention further encompasses anti-ErbB2 antibodies comprising an HCDR3, an LCDR3 or both, three heavy chain CDRs, three light chain CDRs or all six CDRs, a VH or VL or an antigen-binding portion of such a VH or VL. or both, of a specifically provided molecule herein
[0143] Using recombinant DNA methodology, a disclosed CDR sequence may be introduced into a repertoire of VH or VL domains lacking the respective CDR (Marks et al. (BioTechnology (1992) 10: 779-783). For example, a primer adjacent to the 5' end of the variable domain and a primer to the third FR can be used to generate a repertoire of variable domain sequences lacking CDR3. This repertoire can be combined with a CDR3 of an antibody disclosed herein. Using analogous techniques, portions of a disclosed CDR sequence may be shuffled with portions of CDR sequences from other antibodies to provide a repertoire of antigen-binding fragments that bind ErbB2. Either repertoire can be expressed in a host system such as phage display (described in WO 92/01047 and its corresponding U.S. Patent No. 5,969,108) so suitable antigen-binding fragments that bind to ErbB2 can be selected.
[0144] A further alternative uses random mutagenesis of a VH or VL sequence disclosed herein to generate variant VH or VL domains still capable of binding ErbB2. A technique using error-prone PCR is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).
[0145] Another method uses direct mutagenesis of a VH or VL sequence disclosed herein. Such techniques are described by Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91 : 3809-3813) and Schier et al. (J. MoI. Biol. (1996) 263: 551-567).
[0146] Also encompassed by the invention is a portion of a variable domain that comprises at least one CDR region substantially as set out herein and, optionally, intervening framework regions from the VH or VL domains as set out herein. Variable domains lacking a portion of the N-terminus of the FR1 and/or a portion of the Ci terminus of the FR4 are also encompassed by the invention. Additional residues at the N-terminal of the FR1 or C-terminal of the FR4 of the variable domain may not be the same residues found in naturally occurring antibodies. For example, construction of antibodies by recombinant DNA techniques often introduces N- or C-terminal residues from its use of linkers. Some linkers may be used to join variable domains to other variable domains (e.g., diabodies), constant domains, or proteinaceous labels.
[0147] Although the embodiments specifically exemplified herein comprise a "matching" pair of VH and VL domains, a skilled artisan will recognize that alternative embodiments may comprise binding proteins containing only a single CDR from either VL or VH domain. Either one of the VH domain or VL domain can be used to screen for complementary domains capable of forming a two-domain specific binding protein capable of, binding to ErbB2 ECD. The screening may be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO 92/01047. In this approach, an individual colony containing either a H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H), and the resulting two-chain specific antigen-binding domain is selected in accordance with phage display techniques as described. [0148] In some alternative embodiments, the anti-ErbB2 binding protein can be linked to a protein (e.g., albumin) by chemical cross-linking or recombinant methods. The disclosed antibodies may also be linked to a variety of nonproteinaceous polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes) in manners set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192; or 4,179,337. The binding proteins can be chemically modified by covalent conjugation to a polymer, for example, to increase their half-life in blood circulation. Exemplary polymers and attachment methods are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.
[0149] Binding proteins of the invention can be modified to alter their glycosylation; that is, at least one carbohydrate moiety can be deleted or added to the binding protein, for example to modify antibody dependent (or Fc dependent) cellular cytotoxicity (ADCC/FcDCC), in particular to enhance ADCC/FcDCC.
Deletion or addition of glycosylation sites can be accomplished by changing amino acid sequence to delete or create glycosylation consensus sites, that are well known in the art. Another means of adding carbohydrate moieties is the chemical or enzymatic coupling of glycosides to amino acid residues of the antibody (see WO 87/05330 and ApNn et al.
(1981 ) CRC Crit. Rev. Biochem., 22: 259-306). Removal of carbohydrate moieties can also be accomplished chemically or enzymatically (see Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259: 52; Edge et al. (1981 ) Anal. Biochem., 118: 131 ; Thotakura et al. (1987) Meth. Enzymol., 138: 350). [0150] Methods for altering an antibody constant region are known in the art. Antibodies with altered function (e.g., altered affinity for an effector ligand such as FcR on a cell or the C1 component of complement) can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 A1 , US 5,624,821 and US 5,648,260). Similar types of alterations could be described that if applied to a murine or other species antibody would reduce or eliminate similar functions.
[0151] For example, it is possible to alter the affinity of an Fc region of an antibody (e.g., an IgG, such as a human IgG) for FcR (e.g., Fc gamma R1 ) or C1q. The affinity may be altered by replacing at least one specified residue with at least one residue having an appropriate functionality on its side chain, or by introducing a charged functional group, such as glutamate or aspartate, or perhaps an aromatic non-polar residue such as phenylalanine, tyrosine, tryptophan or alanine (see e.g., US 5,624,821).
[0152] For example, replacing residue 297 (asparagine) with alanine in the IgG constant region significantly inhibits recruitment of effector cells, while only slightly reducing (about three fold weaker) affinity for CIq (see e.g., US 5,624,821 ). The numbering of the residues in the heavy chain is that of the EU index (see Kabat et al., 1991 supra). This alteration destroys the glycosylation site and it is believed that the presence of carbohydrate is required for Fc receptor binding. Any other substitution at this site that destroys the glycosylation site is believed to cause a similar decrease in lytic activity. Other amino acid substitutions, e.g., changing any one of residues 318 (GIu), 320 (Lys) and 322 (Lys), to Ala, are also known to abolish CIq binding to the Fc region of IgG antibodies (see e.g., US 5,624,821).
[0153] Modified binding proteins can be produced that have a reduced interaction with an Fc receptor. For example, it has been shown that in human IgG3, which binds to the human Fc gamma R1 receptor, changing Leu 235 to GIu destroys its interaction with the receptor. Mutations on adjacent or close sites in the hinge link region of an antibody (e.g., replacing residues 234, 236 or 237 with Ala) can also be used to affect antibody affinity for the Fc gamma R1 receptor. The numbering of the residues in the heavy chain is based in the EU index (see Kabat et al., 1991 supra).
[0154] Additional methods for altering the lytic activity of an binding protein, for example, by altering at least one amino acid in the N-terminal region of the CH2 domain, are described in WO 94/29351 by Morgan et al. and US 5,624,821. [0155] One of skill in the art will appreciate that the modifications described above are not all-exhaustive, and that many other modifications are obvious to a skilled artisan in light of the teachings of the present disclosure.
[0156] A binding protein of this invention may be tagged with a detectable or functional label. These labels include radiolabels (e.g., 131I or 99Tc), enzymatic labels (e.g., horseradish peroxidase or alkaline phosphatase), and other chemical moieties (e.g., biotin).
[0157] In some embodiments, the invention features a human, monoclonal antibody that specifically binds the ECD, ErbB2, in particular, human ErbB2 and posseses onr or more of the following characteristics: (1) it is an in vitro generated antibody (2) it is an in vivo generated antibody (e.g., transgenic mouse system); (3) it binds to ErbB2 with an association constant of at least 1012 M"1; (4) it binds to ErbB2 with an association constant of at least 1011 M"1; (5) it binds to ErbB2 with an association constant of at least 1010 M"1; (6) it binds to ErbB2 with an association constant of at least 109 M"1; (7) it binds to ErbB2 with an association constant of at least 106 M'1; (8) it binds to ErbB2 with a dissociation constant of 500 nM or less; (9) it binds to ErbB2 with a dissociation constant of 10 nM or less; (10) it binds to ErbB2 with a dissociation constant of 150 pM or less; (11) it binds to ErbB2 with a dissociation constant of 60 pM or less.
III. Nucleic Acids, Cloning and Expression Systems
[0158] In another aspect, the invention provides isolated nucleic acids encoding an anti-ErbB2 binding protein of the invention. The nucleic acids may comprise DNA or RNA, and they may be synthetic (completely or partially) or recombinant (completely or partially). Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T. [0159] The invention also contemplates nucleic acids that comprise a coding sequence for a CDR1 , CDR2 or CDR3, a frame-work sequence (including FR1 , FR2, FR3 and/or FR4), a VH domain, a VL domain, or combinations thereof, as disclosed herein, or a sequence substantially identical thereto (e.g., a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical thereto, or that is capable of hybridizing under stringent conditions to the sequences disclosed).
[0160] In one embodiment, the isolated nucleic acid has a nucleotide sequence encoding a heavy chain variable region and/or a light chain variable region of an anti-ErbB2 binding protein comprising at least one heavy chain CDR or light chain CDR, respectively, chosen from the CDR amino acid sequences found in SEQ ID Nos:1-95, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 ,333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , 353, 355, 357, 359, 361 , 363, 365, 367, 369, 371 , 373, 375, 377, 379, 381 , 383, 385, 387, 389, 391 , 393, 395, 397, 399, 401 , 403, 405, 407, 409, 411 , 413, 415, 417, 419, 421 , 423, 425, 427, 429, 431 , 433, 435, 437, 439, 441 , 443, 445, 447, 449, 451 , 453, 455, 457, 459, 461 , 463, 465, 467, 469, 471 , 473, 475, 477, 479, 481 , 483, 485, 487, 489, 491 , 493, 495, 497, 499, 501 , 503, 507, 509, 511 , 513, 515, 517, 519, 521 , 523, 525, or a sequence encoding a CDR that differs by one or two amino acids from the CDR sequences set forth herein. In some embodiments, the nucleic acid encodes an anti-ErbB2 binding protein comprising one, two, or all 3 heavy chain CDRs, one, two or all 3 light chain CDRs or all 6 CDRS in any of an specifically exemplified antibody.
[0161] The nucleic acid can encode only the light chain or the heavy chain variable region, or can also encode an antibody light or heavy chain constant region, operatively linked to the corresponding variable region. In one embodiment, the light chain variable region is linked to a constant region chosen from a kappa or a lambda constant region. The light chain constant region may also be a human kappa or lambda type. In another embodiment, the heavy chain variable region is linked to a heavy chain constant region of an antibody isotype chosen from IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, and IgE. The heavy chain constant region may be an IgG (e.g., an IgG1) isotype. [0162] The nucleic acid compositions of the present invention, while often in the native sequence (of cDNA or genomic DNA or mixtures thereof) except for modified restriction sites and the like, may be mutated in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired. In particular, nucleotide sequences substantially identical to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
[0163] In one embodiment, the nucleic acid differs (e.g., differs by substitution, insertion, or deletion) from that of the sequences provided (e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid). Also within the invention are ErbB2 binding proteins encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid specifically exemplified herein or to its complement. If necessary for this analysis the sequences should be aligned for maximum homology. "Looped out" sequences from deletions or insertions, or mismatches, are considered differences. The difference may be at a nucleotide(s) encoding a non-essential residue(s), or the difference may be a conservative substitution(s).
[0164] The invention also provides nucleic acid constructs in the form of plasmids, vectors, transcription or expression cassettes, that comprise at least one nucleic acid as described herein as well as a host cell that comprises at least one nucleic acid described herein. Suitable host cells for the expression of a binding protein of the invention well be well known in the art and include mammalian, plant, insects, bacterial or yeast cells.
[0165] Also provided are the methods of making an anti-ErbB2 antibody of the invention that is encoded by the nucleic acid(s) comprising sequence described herein. The method comprises culturing host cells under appropriate conditions to express the protein from the nucleic acid. Following expression and production, the encoded pp may be isolated and/or purified using any suitable technique, then used as appropriate. The method can also include the steps of fusing a nucleic acid encoding a scFv with nucleic acids encoding a Fc portion of an antibody and expressing the fused nucleic acid in a cell. The method can also include a step of germlining.
[0166] Antigen-binding fragments, VH and/or VL domains, and encoding nucleic acid molecules and vectors may be isolated and/or purified from their natural environment, in substantially pure or homogenous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the require function.
[0167] Systems for cloning and expressing polypeptides in a variety of host cells are known in the art. Cells suitable for producing antibodies are described in, for example, Fernandez et al. (1999) Gene Expression Systems, Academic Press, eds. In brief, suitable host cells include mammalian cells, insect cells, plant cells, yeast cells, or prokaryotic cells, e.g., E. coli. Mammalian cells available in the art for heterologous polypeptide expression include lymphocytic cell lines (e.g., NSD), HEK293 cells, Chinese hamster ovary (CHO) cells, COS cells, HeLa cells, baby hamster kidney cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.
[0168] In one embodiment, all or a portion of an anti-ErbB2 antibody selected from S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1 R2C_CS_1 H 12,
S1 R2A_CS_1D3, S1 R3B2_BMV_1 E1 , S1 R3C1_CS_1 D3, S1 R3B2_DP47_1 E8, S1 R3B2_BMV_1G2, S1 R3B2_BMV_1 H5, S1R3C1_CS_1A6, S1 R3B2_DP47_1C9, S1 R3B2_DP47_1 E10, S1 R3C1_CS_1 B10, S1 R3A1_BMV_1 F3, S1 R3B1_BMV_1G11 , S1 R3A1_BMV_1 G4, S1R3B1_BMV_1 H11 , S1 R3A1_CS_1 B9, S1 R3B1_BMV_1 H9, S1 R3A1_CS_1 B10, S1R3B1_BMV_1C12, S1 R3C1_BMV_1 H11 , S1 R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1 R2A_CS_10B8, S1 R3A1_DP47_7A6, S1 R3B2_DP47_2G3, S1 R2B_CS_6H11 ,
S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1 R2A_DP47_3C1 , S1 R3A1_DP47_7B2, S1 R3B2_DP47_4E2, S1 R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1R2A_CS_5A1, S1R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9, S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1 R3A1_CS_7A8, S1 R2A_CS_2C9, S1 R3A1_CS_12D1 , S1 R2A_CS_7D4,
S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1 E11 , S5R2_DP47_1H11, S6R3_CS_1 G5, S6R2_DP47_1 H11 , S5R3_DP47_1 A10, S5R2_DP47_1 D11 , S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1 H9 is expressed in HEK293 or CHO cells. In other embodiments, one or more nucleic acids encoding an anti-ErbB2 binding protein of the invention are placed under the control of a tissue-specific promoter (e.g., a mammary specific promoter) and the antibodies are produced in transgenic animals. For example, the antibodies are secreted into the milk of the transgenic animal, such as a transgenic cow, pig, horse, sheep, goat or rodent. [0169] Suitable vectors may be chosen or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. The vectors may also contain a plasmid or viral backbone. For details, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989). Many established techniques used with vectors, including the manipulation, preparation, mutagenesis, sequencing, and transfection of DNA, are described in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons (1992).
[0170] A nucleic acid encoding all orjpart of an anti-ErbB2 binding protein of the invention may be introduced into a host cell by any readily available means. For eukaryotic cells, suitable transfection techniques may include calcium phosphate, DEΞAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses, e.g., vaccinia or baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. DNA introduction may be followed by a selection method (e.g., drug resistance) to select cells that contain the nucleic acid.
IV. Therapeutic Uses of Anti-ErbB2 Binding Proteins
[0171] Anti-ErbB2 binding proteins of the invention may be ErbB2 agonists or antagonists. An agonist ErbB2 binder of the invention increases HER2 tyrosine phosphorylation in the absence or presence of other HER2 agonists such as Heregulin or Epidermal Growth Factor (EGF). Certain HER2 agonists of the invention increase phosphorylation of HER2 pathway proteins. In some embodiments, the agonist of the invention increase phosphorylation of AKT, MAPK and/or ERK. In some embodiments, the HER2 agonist of the invention decreases proliferation and/or increases cell death of a cancer cell, in vitro and in vivo.
[0172] Anti-ErbB2 binding proteins that act as antagonists to ErbB2 can be used to reduce at least one ErbB2-mediated activity, such as reducing ErbB2-mediated tyrosine phosphorylation, decreased heterodimerization of ErbB2 with other ERBB-family members, decreased ErbB2-mediated cell signalling and decreased growth or proliferation of ErbB2- expressing cells. In one embodiment, anti-ErbB2 binding proteins of the invention are used in a method for decreasing tumor growth, the method comprising contacting an ErbB2 expressing cell with a binding protein of the invention to modulate cell proliferation, cytolytic activity, cytokine secretion, or chemokine secretion.
[0173] Accordingly, the binding proteins of the invention can be used to directly or indirectly inhibit or reduce the activity (e.g., proliferation, differentiation, and/or survival) of cells expressing ErbB2, and, thus, can be used to treat a variety of disorders including hyperproliferative disorders.
[0174] The binding proteins of the invention can be used to treat hyperproliferative disorders associated with activity of ErbB2 by administering the antibodies in an amount sufficient to inhibit or reduce hyperproliferation and/or to increase cell death, such as by apoplosis of ErbB2 expressing cells in a subject and allowing the antibodies to treat or prevent the disorder. ErbB2 is expressed in a number of cancers including, but not limited to, breast, bladder, cervical, ovarian, prostate, testicular, oral, colorectal, lung and pancreatic, cancers and in childhood medulloblastoma, oral squamous cell carcinoma, gastric cancer cholangio carcinoma, osteosarcoma, primary Fallopian tube carcinoma, salivary gland tumors and synovial sarcoma. Binding proteins of the invention may be used to inhibit the progression of neoplasms, e.g. squamous cell carcinomas, basal cell carcinomas, transitional cell papillomas and carcinomas, adenomas, adenocarcinoma. According to the invention, an anti-ErbB2 binding protein of the invention can be administered to a subject in need thereof as part of a regimen that comprises another therapeutic modality, such as surgery or radiation.
V. Combination Therapy
[0175] According to the invention, a composition suitable for pharmaceutical use comprising at least one anti-ErbB2 binding protein further comprises at least one additional therapeutic agent. The therapy is useful for treating ErbB2-mediated pathological conditions or disorders including cancer. The term "in combination" in this context means that the binding protein composition and the additional therapeutic agent are given as part of a treatment regimen. In some embodiments, the anti-ErbB2 binding protein is administered substantially contemporaneously, either simultaneously or sequentially with another therapeutic agent, including one being a pretreatment in relation to the other. In some embodiments, in which administration is sequential, at the onset of administration of the second agent, the first of the two agents is still detectable at effective concentrations at the site of treatment. In another embodiment, if given sequentially, at the onset of administration of the second compound, the first of the two compounds is not detectable at effective concentrations at the site of treatment.
[0176] According to the invention, a treatment regimen may comprise two or more anti-ErbB2 antibodies of the invention. The binding molecules may be ones that bind the same or nearby regions of HER2, as illustrated for example by blocking or cross-blocking each other's binding to HER2, or they may bind to different regions of HER2, as shown by lack of cross-blocking. Two or more anti-ErbB2 binding molecules of the invention may be co-formulated, co-administered or merely be part of the same treatment regimen.
[0177] For example, the combination therapy can include at least one anti-ErbB2 binding protein of the invention co-formulated with, co-administered with, or administered as part of the same therapeutic regimen as at least one additional therapeutic agent. The additional agents may include at least but is not limited to mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics, antiproliferative agents, kinase inhibitors, angiogenesis inhibitors, growth factor inhibitors, cox-l inhibitors, cox-ll inhibitors, radiation, cell cycle inhibitors, enzymes, anti-hormones, statins, and anti-androgens.
[0178] In other embodiments, at least one anti-ErbB2 binding protein can be co- formulated with, and/or co-administered with, at least one anti-inflammatory drug, immunosuppressant, metabolic inhibitor, and enzymatic inhibitor.
[0179] In other embodiments, an anti-ErbB2 antibody can be used in combination with at least one binding protein, such as an antibody, directed at other cancer targets. Another aspect of the present invention accordingly relates to kits for carrying out the administration of the anti-ErbB2 binding protein alone or in combination with other therapeutic agents. In one embodiment, the kit comprises at least one anti-ErbB2 binding protein formulated in a pharmaceutical carrier, and at least one additional therapeutic agent, formulated as appropriate in one or more separate pharmaceutical preparations. [0180] In one embodiment, the present inventive binding proteins can be administered in combination with (e.g., prior to, concurrently with, or subsequent to) one or more other therapeutic agents. Such therapeutic agents include, for example, cytotoxic agents that inhibit or prevent the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. 1131 , 1125, Y90 and Re186), chemotherapeutic agents, growth inhibitory agents, cytokine, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
[0181] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidaniine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhόne-
Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0182] A growth inhibitory agent when used herein refers to a compound or composition that inhibits growth of a cell, especially an ErbB2-overexpressing cancer cell either in vitro or in vivo. In the context of the present invention, the growth inhibitory agent can be one that significantly reduces the percentage of ErbB2 overexpressing cells in S phase and the binding proteins of the present invention may potentially sensitize the cells to such an S phase agent. S-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo Il inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), include agents that induce G1 arrest and M-phase arrest. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1 , entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
[0183] Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor, fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-l and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -Y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1 , IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. [0184] The invention also pertains to immunoconjugates comprising the binding proteins described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Such immunconjugates are particularly indicated for those binding proteins of the invention that internalize in Her2 expressing cells, as shown in the Examples section.
[0185] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated anti-ErbB2 binding proteins. Examples include 212Bi, 1311, 131 In1 90 Y and 186Re.
[0186] Immunoconjugates comprising a member of the potent family of antibacterial and antitumor agents, known collectively as the calicheamicins or the LL-E33288 complex, (see U.S. Pat. No. 4,970,198 (1990)) are also contemplated. The most potent of the calicheamicins is designated v 1 , which is herein referenced simply as gamma. These compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides, at the same time introducing a functional group such as a hydrazide or other functional group that is useful in attaching a calicheamicin derivative to a carrier. (See U.S. Pat. No. 5,053,394). Conjugation methods for preparing monomeric calicheamicin derivative/carrier have been disclosed (see U.S. Pat. No. 5,712,374 and U.S. Pat. No. 5,714,586, incorporated herein in their entirety).
[0.187] Conjugates of the binding protein and cytotoxic agent can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the binding protein.
[0188] Effective amounts of the other therapeutic agents are well known to those skilled in the art. However, it is well within the skilled artisan's purview to determine the other therapeutic agent's optimal effective amount range. The binding proteins of the present invention and the other therapeutic agent(s) can act additively or, alternatively, synergistically. In one embodiment of the invention, where another therapeutic agent(s) is administered to an animal, either the effective amount of the binding protein of the present invention or the other therapeutic agent(s) can be administered in an amount that is less than its effective amount would be where the other therapeutic agent is not administered. In this case, without being bound by theory, it is believed that the two (or more) act synergistically.
VI. Diagnostic Uses [0189] In a further aspect, a binding protein of the invention may also be used to detect the presence of ErbB2 or ErbB2 expressing cells in a biological sample. By correlating the presence or level of ErbB2 with a medical condition, one of skill in the art can diagnose the associated medical condition, including cancer.
[0190] Binding protein-based, including antibody-based detection methods are well known in the art, and include ELISA, radioimmunoassays, immunoblots, Western blots, flow cytometry, immunofluorescence, immunoprecipitation, and other related techniques. The antibodies may be provided in a diagnostic kit that incorporates at least one of these procedures to detect ErbB2. The kit may contain other components, packaging, instructions, or other material to aid the detection of the protein and use of the kit. [0191] Binding proteins of the invention may be modified with detectable markers, including ligand groups (e.g., biotin), fluorophores and chromophores, radioisotopes, electron-dense reagents, or enzymes. Enzymes are detected by their activity. For example, horseradish peroxidase is detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. Other suitable binding partners include biotin and avidin, IgG and protein A1 and other receptor-ligand pairs known in the art.
[0192] Binding proteins of the invention can also be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to at least one other molecular entity, such as another antibody (e.g., a bispecific or a multispecific antibody), toxins, radioisotopes, cytotoxic or cytostatic agents, among others for therapeutic use. Other permutations and possibilities are apparent to those of ordinary skill in the art, and they are considered equivalents within the scope of this invention.
[0193] Further, the anti-ERRB2 binding proteins can be used to detect the presence, isolate, and/or to quantitate ErbB2-expressing cells in a sample from a subject or by in vivo imaging.
VII. Pharmaceutical Compositions and Methods of Administration
[0194] In still another aspect, the invention provides compositions comprising an anti-ErbB2 binding protein of the invention. The compositions may be suitable for pharmaceutical use and administration to patients. The compositions comprise a binding protein of the present invention and a pharmaceutically acceptable carrier. The composition may optionally comprise a pharmaceutical excipient. As used herein, "pharmaceutical excipient" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Use of these agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
[0195] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. Pharmaceutical compositions may be topically or orally administered, or capable of transmission across mucous membranes. Examples of administration of a pharmaceutical composition include oral ingestion or inhalation. Administration may also be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, cutaneous, or transdermal. [0196] Solutions or suspensions used for intradermal or subcutaneous application typically include at least one of the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases. Such preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials. [0197] Solutions or suspensions used for intravenous administration include a carrier such as physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), ethanol, or polyol. In all cases, the composition must be sterile and fluid for easy syringability. Proper fluidity can often be obtained using lecithin or surfactants. The composition must also be stable under the conditions of manufacture and storage. Prevention of microorganisms can be achieved with antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents (sugar), polyalcohols (mannitol and sorbitol), or sodium chloride may be included in the composition. Prolonged absorption of the composition can be accomplished by adding an agent that delays absorption, e.g., aluminum monostearate and gelatin.
[0198] Oral compositions include an inert diluent or edible carrier. The composition can be enclosed in gelatin or compressed into tablets. For the purpose of oral administration, the antibodies can be incorporated with excipients and placed in tablets, troches, or capsules. Pharmaceutically compatible binding agents or adjuvant materials can be included in the composition. The tablets, troches, and capsules, may contain (1 ) a binder such as microcrystalline cellulose, gum tragacanth or gelatin; (2) an excipient such as starch or lactose, (3) a disintegrating agent such as alginic acid, Primogel, or corn starch; (4) a lubricant such as magnesium stearate; (5) a glidant such as colloidal silicon dioxide; or (6) a sweetening agent or a flavoring agent.
[0199] The composition may also be administered by a transmucosal or transdermal route. For example, antibodies that comprise a Fc portion may be capable of crossing mucous membranes in the intestine, mouth, or lungs (via Fc receptors). Transmucosal administration can be accomplished through the use of lozenges, nasal sprays, inhalers, or suppositories. Transdermal administration can also be accomplished through the use of a composition containing ointments, salves, gels, or creams known in the art. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used. For administration by inhalation, the antibodies are delivered in an aerosol spray from a pressured container or dispenser, that contains a propellant (e.g., liquid or gas) or a nebulizer.
[0200] In certain embodiments, the binding proteins of this invention are prepared with carriers to protect against rapid elimination from the body. Biodegradable polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid) are often used. Methods for the preparation of such formulations are known by those skilled in the art. Liposomal suspensions can be used as pharmaceutically acceptable carriers too. The liposomes can be prepared according to established methods known in the art (U.S. Patent No. 4,522,811 ).
[0201] The binding proteins or compositions of the invention are administered in therapeutically effective amounts as described. Therapeutically effective amounts may vary with the subject's age, condition, sex, and severity of medical condition. Appropriate dosage may be determined by a physician based on clinical indications. The binding proteins or compositions may be given as a bolus dose to maximize the circulating levels of protein for the greatest length of time. Continuous infusion may also be used after the bolus dose. [0202] As used herein, the term "subject" is intended to include human and non- human animals. Subjects may include a human patient having a disorder characterized by cells that express ErbB2, e.g., a cancer cell or an immune cell. The term "non-human animals" of the invention includes all vertebrates, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. [0203] Examples of dosage ranges that can be administered to a subject can be chosen from: 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg/kg to 1 mg/kg, 250 μg/kg to 2 mg/kg, 250 μg/kg to 1 mg/kg, 500 μg/kg to 2 mg/kg, 500 μg/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 mg/kg , 10 mg/kg to 25 mg/kg, 15 mg/kg to 25 mg/kg, 20 mg/kg to 25 mg/kg, and 20 mg/kg to 30 mg/kg (or higher). These dosages may be administered daily, weekly, biweekly, monthly, or less frequently, for example, biannually, depending on dosage, method of administration, disorder or symptom(s) to be treated, and individual subject characteristics. Dosages can also be administered via continuous infusion (such as through a pump). The administered dose may also depend on the route of administration. For example, subcutaneous administration may require a higher dosage than intravenous administration.
[0204] In certain circumstances it may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited for the patient. Each dosage unit contains a predetermined quantity of antibody calculated to produce a therapeutic effect in association with the carrier. The dosage unit depends on the characteristics of the antibodies and the particular therapeutic effect to be achieved.
[0205] Toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50IED50. Binding proteins that exhibit large therapeutic indices may be less toxic and/or more therapeutically effective.
[0206] The data obtained from the cell culture assays and animal studies can be used to formulate a dosage range in humans. The dosage of these compounds may lie within the range of circulating antibody concentrations in the blood, that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage composition form employed and the route of administration. For any antibody used in the present invention, the therapeutically effective dose can be estimated initially using cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of antibody that achieves a half-maximal inhibition of symptoms). The effects of any particular dosage can be monitored by a suitable bioassay. Examples of suitable bioassays include DNA replication assays, transcription-based assays and ErbB2 binding assays.
EXAMPLES
Example 1 : Selection of Anti-ErbB2 scFv's
[0207] Single chain fragment variable (scFv) moieties that bind to the extracellular domain (ECD) of Her2 (ErbB2) were identified following three rounds of selection using three phagemid libraries: the Bone Marrow Vaughan (BMV) library (Vaughan et al, 1996), the combined spleen (CS) library and the DP47 library (unpublished). Several Her2-Fc proteins or cell lines expressing various forms of Her2 were used during the selection and subsequent screening steps (see Table 3). The selection strategies are outlined in Figure 1.
Selection using biotinylated HER2 proteins
[0208] For selections involving biotinylated protein, aliquots of phage and magnetic streptavidin beads (Dynabeads M-280 streptavidin) were blocked separately in 3% milk/PBS for 1 hour at room temperature in a rotary mixer (20 rpm). Each selection was preceded by a de-selection step. For de-selection, blocked phage were incubated with the pre-blocked magnetic beads and incubated for one hour on a rotary shaker (20 rpm). The de-selected library was collected by pelleting the beads using a magnetic separator. A 1 μM concentration of a non-biotinylated competitor protein (eg, irrelevant MlgG2a protein) was added to the de-selected phage and incubated for a further hour.
[0209] Biotinylated selection antigen (at various concentrations as indicated in Figure 1 ) was incubated with the de-selected phage library for 2 hours at room temp on a rotary mixer (20 rpm) followed by a 15 minute incubation with pre-blocked magnetic beads. Beads were separated using a magnetic separator and washed 10 times with PBS/0.1% Tween 20 and 3 times with PBS. Bound phage were eluted by incubation with a 10 ug/ml solution of trypsin in PBS for 30 minutes at 37°C (100 rpm) followed by separation from the magnetic beads.
Selection using cells expressing HER2 ECD or ECD fragments
[0210] For selections involving cells, approximately 4 x 107 de-selection cells (ie. cells not expressing the antigen of interest) and 2 x 107 capture (i.e., selection) cells (cells expressing the antigen of interest) were collected using PBS/5 mM EDTA and washed twice with PBS. Cells were blocked with 3% milk/1% BSA/PBS for 1 hour at 4°C on a rotary mixer (20 rpm). De-selection cells were collected by centrifugation, re-suspended in blocked phage and incubated at 4°C as before. Both the capture and de-selection cells were pelleted and the capture cells were resuspended with the de-selected phage supernatant and incubated at 4°C as before. The capture cells were washed three times with cold PBS/0.1 % Tween 20 and three times with cold PBS. Phage were eluted by re-suspending the cells in a 10 μg/ml trypsin solution and incubated for 30 min at 37°C (100 rpm). Eluted phage were harvested in the supernatant following centrifugation of cells. Eluted phage were used to infect 10 ml of an E. coli TG1 culture that had been grown to mid-logarithmic phase (corresponding to an OD60O of ~ 0.5). Bacteria were infected with phage for 1 hour at 37°C with shaking at 150 rpm, concentrated following a centrifugation step and plated on 2X TY agar bioassay plates containing 2% glucose and 100 ug/ml ampicillin (2X TYAG). Various dilutions of E. coli culture infected with either input or output phage were also plated on 2X TYAG agar to determine phage titers. Following overnight growth at 300C, 10 ml of 2X TYAG medium was added to each bioassay plate and the cells were re-suspended by scraping the bacterial lawn. Glycerol was added to this cell suspension to give a final concentration of 17% and stored in aliquots at -800C until further use. To rescue phage for the next round of selection, 100 μl of this cell suspension was used to inoculate 20 ml 2X TYAG medium, that was grown at 370C (300 rpm) to an OD60O of 0.3-0.5. Cells were then super-infected with 3.3 μl of MK13K07 helper phage and incubated at 37°C (150 rpm) for 1 hour. The cells were then centrifuged and the pellet re-suspended in a kanamycin/non- glucose containing medium (2X TY with 50 μg/ml kanamycin and 100 ug/ml ampicillin). This culture was grown overnight at 300C (300 rpm). Phage were harvested in the supernatant following centrifugation and were ready to use in the second and third rounds of selection as described in Figure 1. Table 3:
Name Description Sequence for Her2 region of fusion protein
HerOOδP Full-length extracellular MELAALCRWGLLLALLPPGAASTQVCT
(Synonyms: domain (ECD) of Her2 GTDMKLRLPASPETHLDMLRHLYQGC
ECD; SIIS; expressed with a mlgG2a Fc QWQGNLELTYLPTNASLSFLQDIQEV HER008) tail QGYVLIAHNQVRQVPLQRLRIVRGTQL
FEDNYALAVLDNGDPLNNTTPVTGASP GGLRELQLRSLTEILKGGVLIQRNPQLC YQDTILWKDIFHKNNQLALTLIDTNRSR ACHPCSPMCKGSRCWGESSEDCQSL TRTVCAGGCARCKGPLPTDCCHEQCA AGCTGPKHSDCLACLHFNHSGICELHC PALVTYNTDTFESMPNPEGRYTFGASC VTACPYNYLSTDVGSCTLVCPLHNQEV
TAEDGTQRCEKCSKPCARVCYGLGME
HLREVRAVTSANIQEFAGCKKIFGSLAF
LPESFDGDPASNTAPLQPEQLQVFETL
EEITGYLYISAWPDSLPDLSVFQNLQVI
RGRILHNGAYSLTLQGLGISWLGLRSL
RELGSGLALIHHNTHLCFVHTVPWDQL
FRNPHQALLHTANRPEDECVGEGLAC
HQLCARGHCWGPGPTQCVNCSQFLR
GQECVEECRVLQGLPREYVNARHCLP
CHPECQPQNGSVTCFGPEADQCVACA
HYKDPPFCVARCPSGVKPDLSYMPIW
KFPDEEGACQPCPINCTHSCVDLDDKG
CPAEQRASPLTSIIS
(SEQ ID NO: 242)
Herd 7P Her2 ECD with a deletion in MELAALCRWGLLLALLPPGAASTQVCT
,Q the membrane proximal 9 GTDMKLRLPASPETHLDMLRHLYQGC
(Synonyms: amjnQ adds ssed wjth QWQGNLELTYLPTNASLSFLQDIQEV
M2PΠI 7^ a mlgG2a Fc tail QGYVLIAHNQVRQVPLQRLRIVRGTQL
HER017) s FEDNYALAVLDNGDPLNNTTPVTGASP
GGLRELQLRSLTEILKGGVLIQRNPQLC YQDTILWKDIFHKNNQLALTLIDTNRSR ACHPCSPMCKGSRCWGESSEDCQSL TRTVCAGGCARCKGPLPTDCCHEQCA AGCTGPKHSDCLACLHFNHSGICELHC PALVTYNTDTFESMPNPEGRYTFGASC VTACPYNYLSTDVGSCTLVCPLHNQEV TAEDGTQRCEKCSKPCARVCYGLGME HLREVRAVTSANIQEFAGCKKIFGSLAF LPESFDGDPASNTAPLQPEQLQVFETL EEITGYLYISAWPDSLPDLSVFQNLQVI RGRILHNGAYSLTLQGLGISWLGLRSL RELGSGLALIHHNTHLCFVHTVPWDQL FRNPHQALLHTANRPEDECVGEGLAC HQLCARGHCWGPGPTQCVNCSQFLR GQECVEECRVLQGLPREYVNARHCLP CHPECQPQNGSVTCFGPEADQCVACA HYKDPPFCVARCPSGVKPDLSYMPIW KFPDEEGACQPCPINCTHSCVDLDDKG CPAEQR
(SEQ ID NO: 243)
HerO18P Her2 ECD with a deletion in MELAALCRWGLLLALLPPGAASTQVCT
,. m/mc the CR2 (Domain IV) region GTDMKLRLPASPETHLDMLRHLYQGC (Synonyms. e essed wjth a m| G2a Fc QWQGNLELTYLPTNASLSFLQDIQEV 1 -8- tail QGYVLIAHNQVRQVPLQRLRIVRGTQL
HER018) FEDNYALAVLDNGDPLNNTTPVTGASP
GGLRELQLRSLTEILKGGVLIQRNPQLC YQDTILWKDIFHKNNQLALTLIDTNRSR ACHPCSPMCKGSRCWGESSEDCQSL TRTVCAGGCARCKGPLPTDCCHEQCA AGCTGPKHSDCLACLHFNHSGICELHC PALVTYNTDTFESMPNPEGRYTFGASC
VTACPYNYLSTDVGSCTLVCPLHNQEV
TAEDGTQRCEKCSKPCARVCYGLGME
HLREVRAVTSANIQEFAGCKKIFGSLAF
LPESFDGDPASNTAPLQPEQLQVFETL
EEITGYLYISAWPDSLPDLSVFQNLQVI
RGRILHNGAYSLTLQGLGISWLGLRSL
RELGSGLALIHHNTHLCFVHTVPWDQL
FRNPHQALLHTANRPEDECVGEGLAC
HQLCARGHCWGPGPTQCVNCSQFLR
GQECVEECRVLQGLPREYVNARHCLP
CHPECQPQNGSVTCFGPEADQCVACA
HYKDPPFCVAR
(SEQ ID NO: 244)
HerO54P Domains I (L1) and Il (CR-1 ) MELAALCRWGLLLALLPPGAASTQVCT of Her2 expressed with a GTDMKLRLPASPETHLDMLRHLYQGC
(Synonyms: m|qG2a Fc tail QWQGNLELTYLPTNASLSFLQDIQEV rR QGYVLIAHNQVRQVPLQRLRIVRGTQL
1 FEDNYALAVLDNGDPLNNTTPVTGASP
1.0) GGLRELQLRSLTEILKGGVLIQRNPQLC
YQDTILWKDIFHKNNQLALTLIDTNRSR ACHPCSPMCKGSRCWGESSEDCQSL TRTVCAGGCARCKGPLPTDCCHEQCA AGCTGPKHSDCLACLHFNHSGICELHC PALVTYNTDTFESMPNPEGRYTFGASC VTACPYNYLSTDVGSCTLVCPLHNQEV TAEDGTQRCEKCSKPC
(SEQ ID NO: 245)
Full length MELAALCRWGLLLALLPPGAASTQVCT
HER2 GTDMKLRLPASPETHLDMLRHLYQGC
QWQGNLELTYLPTNASLSFLQDIQEV QGYVLIAHNQVRQVPLQRLRIVRGTQL FEDNYALAVLDNGDPLNNTTPVTGASP GGLRELQLRSLTEILKGGVLIQRNPQLC YQDTILWKDIFHKNNQLALTLIDTNRSR ACHPCSPMCKGSRCWGESSEDCQSL TRTVCAGGCARCKGPLPTDCCHEQCA AGCTGPKHSDCLACLHFNHSGICELHC PALVTYNTDTFESMPNPEGRYTFGASC VTACPYNYLSTDVGSCTLVCPLHNQEV TAEDGTQRCEKCSKPCARVCYGLGME HLREVRAVTSANIQEFAGCKKIFGSLAF LPESFDGDPASNTAPLQPEQLQVFETL EEITGYLYISAWPDSLPDLSVFQNLQVI RGRILHNGAYSLTLQGLGISWLGLRSL RELGSGLALIHHNTHLCFVHTVPWDQL FRNPHQALLHTANRPEDECVGEGLAC HQLCARGHCWGPGPTQCVNCSQFLR GQECVEECRVLQGLPREYVNARHCLP CHPECQPQNGSVTCFGPEADQCVACA
HYKDPPFCVARCPSGVKPDLSYMPIW
KFPDEEGACQPCPINCTHSCVDLDDKG
CPAEQRASPLTSIISAWGILLVWLGW
FGILIKRRQQKIRKYTMRRLLQETELVE
PLTPSGAMPNQAQMRILKETELRKVKV
LGSGAFGTVYKGIWIPDGENVKIPVAIK
VLRENTSPKANKEILDEAYVMAGVGSP
YVSRLLGICLTSTVQLVTQLMPYGCLLD
HVRENRGRLGSQDLLNWCMQIAKGMS
YLEDVRLVHRDLAARNVLVKSPNHVKIT
DFGLARLLDIDETEYHADGGKVPIKWM
ALESILRRRFTHQSDVWSYGVTVWEL
MTFGAKPYDGIPAREIPDLLEKGERLPQ
PPICTIDVYMIMVKCWMIDSECRPRFRE
LVSEFSRMARDPQRFWIQNEDLGPAS
PLDSTFYRSLLEDDDMGDLVDAEEYLV
PQQGFFCPDPAPGAGGMVHHRHRSS
STRSGGGDLTLGLEPSEEEAPRSPLAP
SEGAGSDVFDGDLGMGAAKGLQSLPT
HDPSPLQRYSEDPTVPLPSETDGYVAP
LTCSPQPEYVNQPDVRPQPPSPREGP
LPAARPAGATLERPKTLSPGKNGWKD
VFAFGGAVENPEYLTPQGGAAPQPHP
PPAFSPAFDNLYYWDQDPPERGAPPS
TFKGTPTAENPEYLGLDVPV
(SEQ ID NO: 246)
Example 2: Preparation of phage or crude periplasmic material for use in ELISAs
[0211] ScFvs can be expressed either on the surface of a phage particle or in solution in the bacterial periplasmic space, depending upon the growth conditions used. To induce release of scFv into the periplasm, 96-deepwell plates containing 2X TY media with 0.1 % glucose/100 μg/ml ampicillin were inoculated from thawed glycerol stocks (one clone per well) using the QPix2 Colony picker (Genetix) and grown at 37°C (999rpm) for ~ 4 hours. Cultures were induced with IPTG at a final concentration of 0.02 mM and grown overnight at 3O0C (999 rpm). The contents of the bacterial periplasm (peripreps) were released by osmotic shock. Briefly, plates were centrifuged and pellets were resuspended in 150 μl
HEPES periplasmic buffer (50 mM HEPES, pH7.4/0.5mM EDTA/20% Sucrose), followed by the addition of 150 μl 1 :5 HEPES:water and incubated on ice for 30 minutes. Plates were centrifuged and the scFv-containing supernatant was harvested.
[0212] To prepare phage expressing scFv on their surface, 96-well plates containing 150 μl 2X TY media with 2% glucose/100 μg/ml ampicillin were inoculated from thawed glycerol stocks as described above and grown at 37°C (700 rpm) for ~ 4 hours. 20 μl of a 1 :1000 dilution of helper phage (- 2 x 108 pfu) was added and the plates incubated for a further hour at 37°C (300 rpm). Plates were centrifuged and the media was replaced with a kanamycin/non-glucose containing media (2X TY with 50 μg/ml kanamycin and 100 ug/ml ampicillin). Plates were grown overnight at 300C (700 rpm) and phage were harvested in the supernatant following centrifugation.
[0213] Thirty-one Her2-binding ScFv's were identified by three rounds of screenings as illustrated in Figure 1. These ScFv's specifically bind to the ECD region of Her2. [0214] Among these thirty-one Her2-binding ScFv's, fourteen ScFv's were expressed on the surface of a phage particle for the purpose of screening. These ScFv's are: S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1 R2C_CS_1 H12, S1 R2A_CS_1 D3, S1 R3B2_BMV_1 E1 , S1 R3C1_CS_1 D3, S1 R3B2_DP47_1 E8, S1 R3B2_BMV_1 G2, S1R3B2_BMV_1 H5, S1 R3C1_CS_1A6, S1 R3B2_DP47_1C9, S1 R3B2_DP47_1 E10, and S1 R3C1_CS_1 B10 (Figures 2 and 3).
[0215] The remaining seventeen ScFv's were expressed in bacterial periplasm in soluble form for the purpose of screening: S1 R3A1_BMV_1 F3, S1 R3B1_BMV_1 G11 , S1 R3A1_BMV_1G4, S1R3B1_BMV_1 H11 , S1 R3A1_CS_1 B9, S1 R3B1_BMV_1 H9, S1 R3A1_CS_1 B10, S1 R3B1_BMV_1 C12, S1 R3C1_BMV_1 H11 , S1 R3B1_BMV_1A10, S1 R3A1_CS_1 D11 , S1 R3C1_DP47_1 H1 , S1 R3A1_CS_1 B12, S1 R3B1_BMV_1 H5, S1 R3A1_DP47_1A6, S1 R3B1_DP47_1 E1 , and S1 R3B1_BMV_1A1 (Figures 2 and 3).
Example 3: ELISA to test Her2 protein construct binding by scFvs expressed in the E. coli periplasm, on the surface of phage, or in mammalian cells as Fc fusions [0216] Various Her2-Fc proteins (e.g., HerOOδP, HerO17P, HerO18P, etc.) or a negative control murine lgG2a protein were coated overnight at 4°C on 96-well Nunc Maxisorp at a concentration of 1 ug/ml in PBS. Alternatively, pre-blocked streptavidin- coated plates (Greiner) were coated with biotinylated Her2-Fc proteins for 1 hour at room temperature at a concentration of 1 ug/ml in block buffer (3% skim milk/1% BSA/PBS). Plates were washed three times using PBS and blocked for 1 hour at room temperature in 3% skim milk/1 % BSA/PBS. Phage or peripreps were prepared as described above and were blocked for 1 hour at room temperature in an equal volume of 6% skim milk/1% BSA/PBS. Blocked plates were washed five times with PBS and 50 μl/well of blocked phage or periprep were transferred to the appropriate plates and incubated for 1 hour at room temperature. A 1 ug/ml solution of HERCEPTIN® (trastuzumab) (in blocking buffer) was added to well H12 of each plate to serve as a positive control. Plates were washed five times with PBS prior to the addition of a 1 :250 dilution of anti-myc peroxidase (Roche), a 1 :2500 dilution of anti-M13 peroxidase (Amersham Biosciences) or a 1 :5000 or 1 :1000 dilution of goat anti-human peroxidase (Southern Biotech) secondary antibody to detect bound scFv, phage, HERCEPTIN® (trastuzumab) or SMIP, respectively. Plates were incubated for a further hour at room temperature and washed seven times with PBS. Signal was developed using TMB, the reaction stopped with H2SO4 and the absorbance read at 450 nm on an Envision plate reader (Perkin Elmer). The results of these binding assays are shown in Figure 5. [0217] Alternatively, plates were coated with 1 ug/ml of a SMIP (Her030,
HerO33/HerO67, HerO18) or antibody (Herceptin®, positive control). SMIPs were used to capture 3-fold serial dilution (9-0 μg/ml) of soluble protein sample (see Figure 27). Captured soluble protein was detected using 0.1 mg/ml anti-c-Erb B2/c-Neu (Ab-5) mouse mAb (TA-1 ; binds ECD; Calbiochem) and detected using HRP-conjugated Goat anti-mouse IgG (Fcg Subclass 1 specific; Jackson ImmuonoResearch).
[0218] The results of the SMIP binding assays are shown in Figure 6A-C, Figure 7A-7D, Figure 8, AND Figures 28-30. In Figure 8, the binding of HER018, HER026- HER039, and Herceptin® (trastuzumab) and HER018, to Her2 protein constructs was scored as -, +, ++ or +++; the, while the binding of HER071-HER087 to Her2 protein constructs was scored as a - or +. In Figure 28, the binding of HER SMIPs to Her2 protein constructs was scored as 0, +, ++, or +++, and cross-reactivity and binding domain are shown. Figure 29 is a graphical summary of the results. HER085 bound soluble full length Her2 ectodomain (ECD) (SIIS dimer) but not soluble Her2 EQR (SIIS lacking membrane proximal amino acids ASPLTSIIS). This indicated that HER085 binding domain required "stumpy" amino acids ASPLTSIIS. HER156 and HER169 did not bind soluble full length HER2 ECD (SIIS dimer) which includes the "stumpy" peptide although they bound the synthetic "stumpy" ASPLTSIIS peptide on which they were selected. This suggests that the "stumpy" peptide in Her2 SIIS was not presented in a form recognized by HER156 and HER169, because HER156 and HER169. The results are summarized in the following Table.
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Example 4: ELISA to measure binding of scFvs (expressed in the Periplasm or on the surface of phage) to Her2-expressed cells
[0219] 2 x 104 CHOK1 cells/well were seeded in a 96-well tissue culture plate on Day 1 and incubated at 37°C/5% CO2 for 2-4 days until a confluent monolayer was observed. Cells were washed five times with PBS (+ Ca/Mg ions) and blocked for 1 hour at room temperature with 3% skim milk/1% BSA/PBS (+ Ca/Mg ions). Phage or peripreps were prepared as described above and were blocked for 1 hour at room temperature in an equal volume of 6% skim milk/1% BSA/PBS (+ Ca/Mg ions). Blocked plates were washed five times with PBS (+ Ca/Mg ions) and 50 μl/well of blocked phage or periprep were transferred to the appropriate plates and incubated for 1 hour at room temperature. A 1 ug/ml solution of HERCEPTIN® (trastuzumab) (in blocking buffer) was added to well H12 of each plate to serve as a positive control. Plates were washed five times with PBS (+ Ca/Mg ions) prior to the addition of a 1 :250 dilution of anti-myc peroxidase (Roche), a 1:2500 dilution of anti-M13 peroxidase (Amersham Biosciences) or a 1 :5000 dilution of goat anti- human (Southern Biotech) secondary antibody to detect bound scFv, phage or HERCEPTIN® (trastuzumab) respectively. Plates were incubated for a further hour at room temperature and washed ten times with PBS (+ Ca/Mg ions). Signal was developed using TMB, the reaction stopped with H2SO4 and the absorbance read at 450 nm on an ENVISION plate reader (Perkin Elmer). The results of these binding assays are shown in Figure 5.
[0220] Alternatively, we tested anti-HER2 SMIP binding to cell line includingJIMT-1 , SKBR3, BT474, 22rv1 , MDA-MB-175, MDA-MB-453, MDA-MB-361 (ATCC), MDA-MB-361 (JL), and Ramos (Her27CD20+ control). Controls used included Herceptin® (trastuzumab), Rituxan® (anti-CD20 mAb rituximab), and CD20-SMIP(2LM20-4 SCCP) .
[0221] Each well of a 6 well plate was seeded with 2 x 105 cells and incubated overnight at 370C / 5% CO2. Cells were then treated with antibody or SMIP (at 10 ug/ml final) (in triplicate) and incubated for another 24 or 48 hours. After incubation, the cells were pulsed with 50 uM BrdU (Sigma) for 30 minutes at 370C, the media was removed, and the cells were treated with trypsin (except Ramos) and then 3-3.5 x 105 cells per well were stained in 100 μl Staining Buffer in the presence or absence of a SMIP or antibody one of three different concentrations (ranging from 200 nM to 0.27 nM). The SMIP or antibody treatment was removed and the cells were washed three times with PBS, pH 7.2-7 A with 0.1% TWEEN®-20 (PBS-T). A secondary antibody (5 ug/ml Alexa Fluor 488-conjugated Goat anti-Human IgG; Molecular Probes #A-11013) was then added and incubated for 1-2 hours at room temperature. The secondary antibody was removed and the cells washed again three times with PBS-T. The cells were then fixed in 1% paraformaldehyde in Staining Buffer and analyzed 1 hour to 1 day later.
[0222] The results of these binding assays are shown in Figure 8 and Figures 9A- 9H and are summarized in Figure 31. (In Figure 9E, 0.82 nM HER094 data not collected due to mechanical error.) As shown in the Figures, SMIPs maintain a similar staining pattern regardless of the amount of HER2 on the cell surface and the other ErbB receptors/ligands expressed by the cell lines (relative surface staining for ErbB1 , Her2, Erb3 and production of ligand by cell lines is not shown). In these experiments, the SMIP staining pattern is HER116 > HER067 > HER012 > HER146 > HER094. Additionally, HER116 binding to BT- 474 cells changes the FSC vs SSC profile of BT-474 cells in a manner that suggests clumping.
JIMT-1 binding assay
[0223] To investigate anti-HER2 SMIP binding to JIMT-1 cells we dissociated JIMT- 1 cells with trypsin or non-enzymatic CellStripper™ (cellgro Mediatech #25-056-C1 ). JIMT-1 ErbB2 epitopes may be partially blocked by MUC4 (Peter Nagy, Elza Friedlander, Minna Tanner, Anita I. Kapanen, Kermit L. Carraway, Jorma Isola, and Thomas M. Jovin.
Decreased Accessibility and Lack of Activation of ErbB2 in JIMT-1 , a Herceptin-Resistant, MUC4-Expressing Breast Cancer Cell Line. Cancer Res 65(2): 473-482, 2005). We then washed cells three times after dissociation and stained them in duplicate with 200 nM Antibody/SMIP (200 nM = 22 μg/mL SMIP). We performed secondary staining with 5 μg/mL Alexa Fluor 488-conjugated Goat anti-Human IgG (Molecular Probes #A-11013).
[0224] The results are shown in Figure 32. As shown in Figure 32, HER085, HER156 and HER169, all "stumpy" binders bind to multiple cell lines harvested by non- enzymatic means better thatn to trypsinized cells, suggesting that the epitope recognized by these SMIPS may be sensitive to trypsin whereas other SMIPs and HERCEPTIN® showed equal or better binding on trypsinized cells. This sensitivity was not likely to be direct as trypsin did not affect the ability of other SMIPs to bind to the full length Her2 ECD (which contains the "stumpy" peptide ASPLTSIIS). The sensitivity may be due to trypsin cleavage of other molecules that are needed for the presentation/exposure of the "stumpy" peptide or the maintenance of Her2 p95 ("stumpy") on the cell surface. Enymatic vs non-enzvmatic preparation
[0225] We further investigated the effects of enzymatic vs non-enzymatic preparation of the cells on SMIP binding. Briefly, we removed media from cells and rinsed them with PBS (-Ca/Mg). We then dissociated the cells with trypsin (0.25%) or non- enzymatic CellStripper™ (Cellgro Mediatech #25-056-C1 ). We washed dissociated cells once in cold media (containing FBS), and washed them again in cold PBS (+Ca/Mg). We then resuspended the cells in cold Staining Buffer (PBS, +Ca/Mg, 2% FBS) and counted resuspended cells. We stained 300,000-350,000 cells per well on ice in 100 μl Staining Buffer +/- primary at 200 nM SMIP/Antibody (200 nM = 22 μg/ml SMIP) with 1 μg/ml Pl. We then washed the stained cells three times with cold Staining Buffer. We performed secondary staining with 5 μg/ml Alexa Fluor 488-conjugated Goat anti-Human IgG (Molecular Probes #A-11013) with 1 μg/ml Pl. We then fixed the cells in 1% Paraformaldehyde in Staining Buffer and analyzed the next day. Results of various cell line staining with "stumpy binders" HER 156 and HER169 are shown in Figure 33. Species cross-reactivity [0226] To determine species cross-reactivity, we performed the above-mentioned binding assay with NIH/3T3 cells transfected with Macaca Her2 and with CHO cells expressing Murine Her2. The results of the cross-reactivity binding studies are summarized in Figure 34. Herceptin®, HER018, HER095, and HER124 cross-react with Macaca Her2, while HER102, HER116, HER133, and HER146 cross-react with both Macaca Her2 and Murine Her2.
Example 5: PCR amplification of scFv regions for sequencing analysis
[0227] PCR amplification of scFvs was carried out using the KOD HOT START DNA Polymerase kit (Novagen) in accordance with the manufacturers instructions. 0.2 μM each of the M13rev (5' G GAAACAG CTATG ACC ATG A 3') (SEQ ID NO: 247) forward and Mycseq (5' CTCTTCTGAGATGAG I I I I I G 3') (SEQ ID NO: 248) reverse primers were used. 5 μl of a 1:10 dilution of a stationary phase bacterial culture was used as the template for a final reaction volume of 20 μl. The cycling conditions used were a 2 minute hot start at 94 0C, 25 cycles of denaturation at 94 0C (1 minute), primer annealing at 42 0C (30 seconds) and extension at 72 0C (1 min), followed by a final 5 minute extension at 72 0C. PCR products were verified by agarose gel electrophoresis and cleaned up with Exol/SAP (shrimp alkaline phosphatase) prior to sequencing of both strands with primers 145837 (51 GGAGATTTTCAACGTGAA 3') (SEQ ID NO: 249) and 142051 (5'
CTCTTCTGAGATGAG I I I I I G 3') (SEQ ID NO: 250). The closest human germlines of the VH and VL segments were determined (Table 4).
Table 4: VH and VL germlines of ERBB2 clones
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Example 6: BIACORE® Binding Assays
[0226] Binding of different Her2-directed binders (antibodies and SMIPs) to monomeric Her2 ECD and truncations of dimeric Her2 ECD were determined using a BIACORE® T100 instrument (GE Healthcare, Biacore, Piscataway, NJ). We conducted the binding experiments in both orientations, i.e., first using anti-HER2 SMIPS as ligands and then as analytes.
SMIPs as ligands
[0229] Her2-directed binders were captured on a chip by a monoclonal mouse anti-human Fc (GE healthcare), which was covalently conjugated to a carboxylmethyl dextran surface (CM4) via amines using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride and Λ/-hydroxysuccinimide. The unoccupied sites of the activated surface were blocked by ethanolamine. The capturing antibody (referred to as anti hFc) binds to the CH2 domain of IgG Fc of all sub-classes and showed no discernible dissociation from the captured her2-binders during the course of the assay. Every cycle, 3 different Her2 binders and a non-binder (negative control) were individually captured by anti hFc on 4 different flow cells, typically to about 50 RU, followed by injection of the analyte (Her2 dimers and monomer) at a particular concentration for 10 minutes over all flow cells. The dissociation of the formed complexes were subsequently followed for 12 minutes. At the end of the cycle, the surface was regenerated gently using 3M MgCI2 which dissociates protein bound to the capturing anti hFc antibody. Multiple such cycles were performed to study binding of different analytes at different concentrations, in the range of 0 - 300 nM, for each set of three Her2 binders captured. Her2 binders were reproducibly captured every cycle with CV not exceeding 1%. The binding was performed at 25 0C in 0.01 M HEPES pH 7.4, 0.15 M NaCI1 0.005% v/v SURFACTANT P20. Signal associated with binding to the negative control was used to subtract for bulk refractive changes. The kinetic parameters and affinities were determined using BIAEVALUATION software. SMIPS as analvtes [0230] In these experiments, the trastuzumab (HERCEPTIN®) and and anti-HER2 SMIPs were used as the analytes and the soluble HER2 receptors were used as the ligands. In one experiment, SMIPs and trastuzumab were flowed over a histidine-tagged monomeric HER2 receptor that was bound to a Ni2+-nitrilotriacetic acid surface. In a second experiment, SMIPs and trastuzumab were flowed over a histidine-tagged HER2 receptor that was captured by an anti-6-histidine-tagged monoclonal antibody conjugated to a CM4 surface. In a third experiment, SMIPs and trastuzumab were flowed over a HER2 receptor that was directly amine-coupled to a CM4 surface. The binding in each of these three experimental designs was performed at 25 0C. When employing any of the three experimental designs listed above, similar results were obtained. Signal associated with binding to the negative control was used to subtract for bulk refractive changes. The kinetic parameters and affinities were determined using BIAEVALUATION software. [0231] The results of the BIACORE® assays are shown in Figures 7 and 35 and in Table 5. HERCEPTIN® (trastuzumab) and the HERCEPTIN® SMIP bound monomeric and dimeric HER2 receptors similarly in both orientations of the BIACORE® assay. HER095 bound monomeric HER2 at sub-nanomolar affinity. In contrast, the HER067, HER033, HER030/HER094, HER 146, HER116 and HER102 SMIPS bound more strongly to dimeric soluble HER2 recpetor than to monomeric HER2 receptor. The HER033 and HER067 SMIPs have the same amino acid sequence, but the difference between them is that the former is produced in HEK cells while the latter is produced in CHO cells. Binding by HER033 and HER067 SMIPs is substantially the same. HER030 appears to bind less strongly than HerO33/HerO67 to the dimers. Specificity for dimeric HER2 may be advantageous in that such binders may have increased selectivity for tumors and may not bind, or show reduced binding to tissues that express low levels of HER2 and/or where ligand independent homodimer formation is limited. Such HER2 binders with reduced binding to non-tumor target tissues (e.g., cardiac tissues) may, thus, have fewer side effects including lower toxicity. In addition, a lack of binding to shed HER2 ectodomain would reduce the effective dose compared to a HER2-binding agent that has significant binding to shed ECD.
Table 5. BIACORE® Affinity Data
Figure imgf000072_0001
NB - No Binding Observed ND - not enough binding to fit
Example 7: BrdU and ATP proliferation assays
[0232] To 96-well plates, cells were added at 2.5 x 103 cells/well (SKBR3, BT474, MDA-MB-453, MDA-MB-175) or at 5 x 103 cells/well (MDA-MB-361 ). The next day, SMIPs were added to the cells at the desired concentration and then incubated at 37°C / 5% CO2 for 4 (SKBR3, MDA-MB-453, MDA-MB-361 , MDA-MB-175), 5 (BT474), or 7 (MDA-MB-361 ) days. The day before cells were harvested, 5-bromo-2'-deoxyuridine (BrdU) is added to a final concentration of 0.1 mM and continued to incubate overnight at 370C. After incubation, media was removed and then the cells were treated with ethanol-based fix solution (DELFIA® Cell Proliferation Kit, Perkin Elmer, Waltham, MA) at room temperature (RT) for 30 minutes. Fix solution was removed by aspiration, 100 μl/well anti-BrdU-Eu labeled antibody (0.5 mg/mL) was added, and the cells were incubated at RT for 2 hours. Cells were then washed 4 times with Tris-based DELFIA Platewash (300 μl/well/wash). DELFIA Inducer (with Triton® X-100, glycine, HCI1 and chelator) was then added to the cells (200 μl/well) and incubated with shaking for 15 minutes at RT. Fluorescence was measured using Flex Station® 3 in Time resolved fluorescence mode (Molecular Devices, Sunnyvale, CA).
[0233] After the proliferation assay fluorescence reading, the DELFIA Inducer was removed by aspiration and Hoechst 33342 nuclear stain solution (Invitrogen, Carlsbad, CA) was added to the cells. Nuclear stain fluorescence was measured on an IN Cell Analyzer at 4x resolution.
[0234] Alternatively, we investigated anti-Her2 SMIP anti-proliferation activity in MDA-MB-361 cells as follows. MDA-MB-361 breast cancer cells were plated in 96-well format and treated with anti-Her2 or control reagents for indicated concentrations and times (24-96hr). For proliferation assays, media (DMEM plus 10% FBS) was removed, the cells washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde and nuclei stained with DAPI (Molecular Probes). Stained nuclei were counted using Cellomics High Content assay measuring fluorescence at 36OnM. The results are shown in Figure 38. For apoptosis assay, fixed cells were permeabilized by treatment with 0.2% Triton 100 in PBS prior to primary staining with mouse anti-cleaved PARP antibody (Cell Signaling Technologies) and secondary staining with goat anti-mouse IgG labeled with ALEXA488 (Invitrogen). Fluorescence was measured in Cellomics High Content assay at 488nM. [0235] ATP Lite First Step assay (Perkin Elmer) was used to assess cellular viability by measuring ATP levels via luminescence (ATP luciferase). To 96-well plates, cells were added at 2.5 x 103 cells/well (SKBR3, BT474, MDA-MB-453, MDA-MB-175) or at 5 x 103 cells/well (MDA-MB-361 ). The next day, SMIPs were added to the cells at the desired concentration and then incubated at 370C / 5% CO2 for 4 (SKBR3, MDA-MB-453, MDA-MB- 361 , MDA-MB-175), 5 (BT474), or 7 (MDA-MB-361 ) days. After SMIP incubation for the desired amount of time, lyophilized ATP Lite substrate is reconstituted with 10 ml of ATP Lite substrate/lysis solution and allowed to sit at room temperature for 10 minutes. This reconstituted substrate solution was added to the cells (100 μl/well) and read luminescence on Top Count Reader (Packard).
[0236] The results of the proliferation assays are shown in Figures 10-12 and Figures 36-38 and are summarized in Figure 39.. As shown in the Figures, the anti-HER2 SMIPS represent different groups of HER2 binders that bind different domains of HER2 and having differential ability to decrease proliferation in multiple cell lines. As shown, anti- HER2 SMIPS reduce proliferation of a different repertoire of cell lines than HERCEPTIN®, the SMIP form of HERCEPTIN® has a different repertoire of cell killing than the parent antibody and HER2 SMIPS differ from each other in the cell lines in which they reduce proliferation.
Example 8: Pathway Phosphorylation Assays
[0237] To 96-well plates, cells were added at 8-12 x 103 cells/well depending on cell type (Becton-Dickinson, San Jose, CA) and allowed to incubate overnight in growth medium with serum at 370C / 5% CO2. After removal of growth medium, the cells were washed with serum-free medium, aspirated, and then serum-free media was added for incubation at 370C / 5% CO2 for 3 hours. The SMIP of interest was prepared in prewarmed serum-free media, added to each well at the indicated concentration, and incubated at 370C / 5%C02 for desired time points. As a control, signaling was inhibited with AG825 (Calbiochem, LaJoIIa, CA) at 40 μM; LY294002 (Cell Signaling) at 50 μM; or U0126 MEK1/2 inhibitor (Cell Signaling) at 10 μM. The cells were then fixed in formaldehyde (diluted in 1x PBS) at a final concentration of 3.7% for 10 minutes at 370C / 5%C02. The cells were then washed two times with PBS. After removing the PBS, the cells were permeabilized in 0.1% Triton® X-100 (Sigma-Aldrich, St. Louis, MO) solution diluted in 1x PBS at room temperature for 5 minutes. The cells were then washed two times with PBS and blocked by incubation in PBS / 1%BSA (Sigma-Aldrich) at room temperature for 30 minutes (or overnight at 40C).
[0238] The blocking solution was removed and primary antibody (in PBS with 3% horse serum or PBS with 1%BSA, and 0.1% Triton® X-100) was added for 1 hour at room temperature (or overnight at 40C). The primary antibodies used (at 0.125 μg/well) were (1 ) rabbit anti-phospho-akt (Ser473) (Cell Signaling, Danvers, MA); (2) mouse anti-phospho- Erk1/2 (Cell Signaling, Danvers, MA); and (3) rabbit anti-phospho-ErbB2 (Abgent, San Diego, CA). The primary antibody was removed and the cells were washed 3 times with PBS. The secondary antibody (in PBS with 3% horse serum or PBS with 1% BSA, and 0.1% Triton® X-100) was then added for 1 hour at room temperature (or overnight at 40C) protected from light. The secondary antibodies used (at 0.2 μg/well) were Alexa 488 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA) and DyLight 649 goat anti-ms IgG (Pierce, Rockford, IL). The secondary antibody was removed and the cells were washed 3 times with PBS. Then 100 μl_ of PBS containing 200 ng/ml Hoechst 33342 nuclear stain (Invitrogen, H3570) (and if needed 1 ug/ml CellMask Blue cytoplasmic stain (Invitrogen, H34558) was added to the cells. The plates were covered and kept protected from light. The plates were then imaged.
[0239] Alternatively, we investigated anti-Her2 SMIP signal transduction activity in MDA-MB-361 cells as follows. MDA-MB-361 breast cancer cells, were plated in 6-well plate to 80-90% confluency (DMEM plus 10% FBS) and treated with anti-Her2 or control reagents for 24hr with and without pretreatment with Heregulin (HRG - 15 min.) or EGF (30 min.). For assay of total and phosphorylated Her2, cells were lysed, 50ug total protein was fractionated using SDS-PAGE and transferred to nitrocellulose membranes using standard procedures. Western blot analysis used either rabbit anti-Her2 antibody (Cell Signaling Technologies), anti-pHer2_Y1248 (Upstate) or anti-Actin (Santa Cruz) as primary antibody and subsequently stained with HRP-conjugated anti-rabbit IgG. Peroxidase activity was measured using ECLplus2 kit (GE Healthcare) following manufacturer's protocols and exposed to film. As shown in Figure 13, HER033 induces HER2 phosphorylation.
[0240] To measure increased downstream phosphoprotein signal transduction, MDA-MB-361 breast cancer cells were plated in 96-well format and treated with anti-Her2 or control reagents for the concentrations and times (10min to 24hr) shown in Figure 15. Media was removed, cells washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton 100/PBS. Cells were subsequently stained with either rabbit anti-pAKT (Cell Signaling Technologies), anti-pERK (Cellomics), anti-pS6K (Cell Signaling Technologies), or anti-p38MAPK (Cell Signaling Technologies). Following PBS wash (3X), cells were stained with secondary goat anti-rabbit IgG antibody labeled with ALEXA594. Cell fluorescence was quantified using Cellomics High Content assay at 594nM.
[0241] HerO67 (HerO33) has agonistic activity (increased signaling) compared to trastuzumab (see Table 6). Moreover, HerO67 and HerO18 are generally a stronger inducer of Her2, Erk1/2, and Akt phosphorylation than trastuzumab. The increase was statistically significant as compared to the mock treatment when measured by the pairwise student T- test (<0.001 ). Table 6. Induction of phosphorylation by HER018, HER067, Herceptin and Heregulin
Figure imgf000076_0001
[0242] We next investigated the effect of inhibiting kinase activity on SMIP anti- proliferative activity. Briefly, we seeded MDA-MB-361 breast cancer cells into 96-well plate format and grew them 48hr in DMEM media plus 10% FBS. We then treated the cells either with 0.3ug/ml of Her146 SMIP or vehicle (control). We additionally co-treated both Her146 and vehicle treated cells with the indicated dose of kinase inhibitor (for MEK:CL-1040 (PD184352); and for ERK1/2: FR180204). Cells were grown an additional 90hr, media removed and fixed (4% paraformaldehyde) and stained with DAPI according to manufacture's protocol (Molecular Probes) and nuclei of viable cells counted using Cellomics High Content assay.
[0243] The results are shown in Figures 40 and 41. Her146 mediated antiproliferative activity is demonstrated by decrease in viable cell count in absence of co- treatment with the kinase inhibitor. Inhibition of MEK with small molecule kinase inhibitor CL- 1040 between 0.4 and 3.7uM demonstrate dose dependent reversal of the Her146 mediated anti-proliferative activity, demonstrating that Her146 activity is mediated by hyperactivation of MEK kinase pathway activity. Higher doses of CL-1040 inhibited cell proliferation by complete inhibition of MEK kinase activity. Similarly, inhibition of ERK1/2 activity (downstream target of MEK) with small molecule kinase inhibitor FR180204 demonstrates dose dependent reversal of the HeM 46 mediated anti-proliferative activity, demonstrating that HeM 46 anti-proliferative activity is mediated by hyperactivation of MEK/ERK pathway.
[0244] We also used siRNA against ERK1 or ERK2 to investigate the effect on SMIP anti-proliferative activity in MDA-MB-361 breast cancer cells. Briefly, the cells were reversed transfected with siRNA oligos (25nM) targeting ERK1 or ERK2 kinases, or with non-targeting control oligo (NTO) using Dharmafect 4 lipid and following manufacture's recommended protocols in 96-well plate format. Cells were grown 60hr in DMEM media plus 10% FBS and then treated with either HeM 46 (0.3ug/ml) or vehicle control as indicated. Cells were then grown an additional 36hr and media removed and cells fixed (4% paraformaldehyde) and stained with DAPI according to manufacture's protocol (Molecular Probes) and nuclei of viable cells counted using Cellomics High Content assay. Viable cell counts from two individual experiments are shown graphically in Figure 42 as open or hatched bars. Treatment of MDA-MB-361 cells with HeM 46 in the presence of non-targeting siRNA (NTO) resulted in inhibition of cell proliferation. siRNA mediated knockdown of ERK1 kinase did not substantially alter the Her146 mediated antiproliferative activity. In contrast, siRNA mediated knockdown of ERK2 kinase significantly reversed the Her146 antiproliferative activity. These data are consistent with reversal of Her146 anti-proliferative activity mediated by pharmacological inhibition of MEK or ERK1/2 and support the conclusion that these are the result of on-target activities. In addition, the results indicate that the hyperactivation of ERK2 but not ERK1 is responsible for cellular anti-proliferative activity of Her146. [0245] We further investigated the duration of HER2 phosphorylation by anti-HER2
SMIP. Briefly, MDA-MB-361 breast cancer cells were grown in DMEM media supplemented with 10% FBS. Cells were treated with either anti-Her2 SMIPs (HerO33, Her146), Herceptin or controls anti-CD20 SMIP or untreated. Additionally, cell populations were either treated with heregulin (HRG1 ), the ligand activator of Her3, or vehicle for a total of 24 or 48hr. Cells were harvested and protein lysates size fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Protein blots were probed with anti-pHer2 (Upstate), anti-pHer3 (Cell Signaling Technologies) or anti-Actin (Santa Cruz, loading control) monoclonal antibodies. Blots were subsequently stained with goat anti-rabbit lgG_coupled to horseradish peroxidase (Santa Cruz) and visualized by ECL staining (GE) following manufacture's protocol. As shown in Figure 43, HerO146 mediates long term (48hr) hyperactivation of Her2 phosphorylation in MDA-MB-361 breast cancer cells.
Example 9: Cell Cycle Assay
[0246] To investigate the effect of the ErbB2 ECD binder on cell cycle in HERCEPTIN® sensitive and HERCEPTIN® resistant cells, each well of a 6 well plate was seeded with 2 x 105 cells (SKBR3 or BT474 (sensitive) or MDA-MB-453 or MDA-MB-361 (resistant)) and incubated overnight at 370C / 5% CO2. Cells were then treated with antibody or SMIP (at 10 μg/ml final) (in triplicate) and incubated for another 24 or 48 hours. After incubation, the cells were pulsed with 50 uM BrdU (Sigma) for 30 minutes at 370C, the media was removed, and the cells were treated with trypsin and harvested in a FACS tube on ice. The cells were washed with PBS, fixed with 70% cold ethanol, and incubated on ice for 30 minutes. The ethanol was removed and then 2N HCI/0.5%Triton X-100 was added, and the cells were incubated for 30 minutes at room temperature (RT). The acid was removed and neutralized with 0.1 M Na2B4O7 for 15 min at RT. The neutralization buffer was removed, FITC labeled anti-BrdU antibody was added (BD Bioscience) in PBS/0.5% TWEEN® 20/1% BSA, and the cells were incubated for 30 minutes at RT in the dark. The FITC dye was removed, the cells washed, and then DAPI nuclear stain (Invitrogen) and RNAse A (Qiagen) each at 1 :1000 dilution was added and the cells were incubated 15 minutes in the dark and then analyzed by FACS. Statistical analysis of the data was performed using ANOVA and Student's t-test.
[0247] The results are presented in Figures 17 and 18. We observed an increased number of cells in the G1 phase in HERCEPTIN® treated SKBR3, BT474 and MDA-MB-453 cells. Among cells treated with HER033 SMIP1 we observed an increased number of cells in S phase in SKBR3 and BT474 cells. [0248] Additional results are presented in Figures 44A-B and 45A-E. Our results demonstrated that SMIPs have different effects on the cell cycle than Herceptin. While both Herceptin and SMIPs inhibited proliferation in SKBR3 and BT474 cells after 24 hours, Herceptin induced G1 arrest and SMIPs induced S-phase arrest. Additionally, while Herceptin did not inhibit cell cycle progression in MDA-MB-453, MDA-MB-361 (JL), and MDA-MB-361 (ATCC) cells after 24-48 hours, SMIP treatment inhibited the cell cycle by inducing G1 arrest. The cell cycle inhibition was not observed in MDA-MB-361 (ATCC) until 48 hours, but these cells grow slower than the other cell lines. Finally, HER116 appeared to behave a little differently than HER030/094, HER033/067, and HER146.
[0249] Specifically, we observed a decreased number of cells in the G1 phase in HER033, HER067, HER102, HER122 and Heregulin treated SKBR3 cells and in HER033,
HER067, HER146, HER102, HER122 and Heregulin treated BT474 cells. We also observed an increased number of cells in the G1 phase in Herceptin® treated SKBR3 and BT474 cells; HER033, HER067, HER146, and HER116 treated MDA-MB-453 cells at 24 hours; HER033, HER067, and HER146 treated MDA-MB-361 (JL) cells at 24 hours; HER094, HER067, and HER146 treated MDA-MB-361 (JL) cells at 48 hours; Herceptin treated MDA- MB-361 (ATCC) cells at 24 hours; and HER094, HER067, and HER146 treated MDA-MB- 361 (ATCC) cells at 48 hours.
[0250] Treatment with HER094, HER0333, HER067, HER146, HER116, HER124, and Heregulin resulted in an increase in the number of SKBR3 cells in S-phase at 24 hours. Treatment with HER018, HER094, HER033, HER067, HER146, HER116, HER102,
HER124, and heregulin increased the number of BT474 cells in S-phase at 24 hours. We also observed an increase the number of cells in S-phase in Heregulin treated MDA-MB-361 (JL) cells at 24 hours; and HER018 and Heregulin treated MDA-MB-361 (ATCC) cells at 48 hours. [0251] We observed a decreased number of cells in S-phase in Herceptin® treated SKBR3 and BT474 cells; HER067 treated MDA-MB-453 cells; HER033 and HER067 treated MDA-MB-361 (JL) cells at 24 hours; HER094, HER033, HER067 and HER146 treated MDA- MB-361 (JL) cells at 48 hours; and HER146 treated MDA-MB-361 (ATCC) cells at 48 hours. [0252] HER067, HER146, and HER116 treatment decreased the number of SKBR3 cells in G2M phase. HER018, HER094, HER033, HER146, HER116, HER102, and heregulin decreased the number of BT474 in G2M phase. Alternatively, out of the Herceptin-resistant cell lines, only MDA-MB-361 (ATCC) cells at 48 hours showed significantly decreased G2M phase cells following SMIP treatment (HER094, HER067, HER146 and heregulin).
Example 10 In Vivo Xenograft Assay
[0253] To investigate the effect of the ErbB2 binding molecules of the invention in vivo, we tested the molecules in three mouse models.
SCID/Beige Mouse Model
[0254] Female (6-7 week old) Beige SCID mice (Beige SCID CB-17/lcrHsd- Prkdcscid-Lystbg) were obtained from Harlan Sprague Dawley, NJ. Virus free MDA-MB-361 cells were thawed from a new vial and cultured to generate appropriate numbers. Cells were grown to near confluency and had a viability of >90%. Cells were harvested, washed twice with sterile PBS, resuspended to 2 x 108 cells/ml, then combined with Matrigel 1 :2. and kept on ice until injection.
[0255] Tumor Cell Implantation and Monitoring: Each mouse was injected with 100 μl of the cell/Matrigel suspension (1 x 1O7 CeIIs ) subcutaneously on the right flank. Mice were monitored daily for tumor growth. Tumors were established when they reached about 150 to about 300 mm3 (Volume = 1Λ[length x (width)2). Tumors developed in 100% of the implanted mice. Mice were sorted into groups according to tumor size, keeping means consistent among groups using LabCat software. Sorting occurred on day 0, which was the same day the mice received their first treatment.
[0256] Mice were monitored (i.e., weighed and tumors measured) two to three times weekly. Mice were sacrificed if ulceration of tumor occurred, extreme body weight loss (greater than or equal 20%), tumor exceeded about 1200 to about 1500 mm3, or tumor inhibited mobility of a mouse. The study is continued for a total of about 60 days. [0257] Treatment: Mice were sorted into three groups of 11 mice each. Treatment began on day 0 (about six days after cell implantation). Each mouse of a group received intraperitoneal treatments twice a week (for a total of five treatments), which were given in equimolar amounts (900 nM) of (1) SMIP HER067 (100 μg), (2) Herceptin (136 μg, positive control), or (3) human IgG (136 μg, negative control). Survival and tumor size was recorded two to three times weekly. Results were graphed (+/- SEM) and analyzed using Prism software (see Figures 21 and 22).
[0258] In a subsequent experiment, mice were sorted into 4 groups: (1 ) HER146 (100 μg), (2) HER116 (100 μg), (3) Herceptin (136 μg, positive control) and (4) human IgG (136 μg, negative control). Survival and tumor size was recorded two to three times a week. Results were graphed (+/- SEM) and analyzed using Prism software (see Figures 46 and 47)
BALB/c nu and nu/nu mouse models
[0259] Male BALB/c nu/nu (nude) mice (18-23 g) and female nu/nu (nude) mice (18-23 g) were obtained from Charles River Laboratories, Wilmington, MA.
Subcutaneous BCL xenografts:
[0260] Female, athymic nude mice were exposed to total body irradiation (400 rads) to further suppress their residual immune system and facilitate the establishment of xenografts. Three days later, the irradiated mice were injected subcutaneously (SC) with 1x107 MDA-MB-361 cells in Matrigel (Collaborative Biomedical Products, Belford, MA, diluted 1 :1 in culture medium) in the dorsal, right flank. When the tumors reached the mass of 0.1 to 0.25 g, the tumors were staged to ensure uniformity of the treatment groups. Male, athymic Balb/c nude mice were injected s.c. with 1x107 cells in the right flank. When tumors reached an average tumor mass of of 0.1 to 0.25 g, the tumors were staged to ensure uniformity of the treatment groups. Mice were dosed with compounds (100 μg/mouse ip) on days 1 ,4,6,8 and 11 (n=10 mice/treatment group). All compounds were administered ip. Tumors were measured at least once a week and their mass (± SEM) was calculated. Tumor mass for each treatment group was compared to that from the vehicle-treated group for statistical significance using ANOVA and subsequent pairwise comparisons to the vehicle-treated group using a one-tailed t-test with the error term for the t-test based on the pooled variance across all treatment groups. The results are shown in Figures 19, 20, 48A- D and 49A-D.
[0261] The preliminary results in vivo as shown in Figures 46 and 47, however, indicate that HER116 was not efficacious against MDA-MB-361 (JL) xenografts in SCID- Beige mice and did not improve survival compared to negative control. These data correlate with other data herein that show that HER116 appears to lack in vitro anti-proliferation activity against MDA-MB-361 (JL) cells. HER146, on the other hand, was efficacious against MDA-MB-361 (JL) xenografts in SCID-Beige mice but demonstrated slower tumor regression than the positive control. Despite slower tumor regression, HER146 treated mice had better overall survival and tumor free progression than Herceptin (positive control) treated mice. [0262] Based on the in vitro and in vivo results taken as a whole, the anti-ErbB2 binding proteins are believed to be efficacious in treating tumors.
Example 11 : Identification and screening of antibodies that bind to the membrane proximal region of Her2/ERBB2
[0263] Ligand binding triggers ERBB2 dimerization and the activation of the intracellular kinase domain of ERBB2. Autophosphorylation of C-terminal tyrosines triggers the recruitment to these sites of intracellular signal transducers that regulate cellular processes such as proliferation, differentiation, motility, adhesion, protection from apoptosis, and transformation.
[0264] ERBB2 is frequently over-expressed in breast cancer. The existence of high levels of circulating soluble ERBB2 extracellular domain is associated with poor prognosis and decreased responsiveness to chemotherapy and endocrine therapy. In cell cultures, it has been shown that soluble ERBB2 extracellular domain arises by proteolytic cleavage of the extracellular domain of ERBB2. The cleavage of the extracellular domain results in a truncated, cell-associated, ERBB2 fragment that contains the intracellular kinase domain and a potentially surface-exposed N-terminal membrane proximal sequence, EQRASPLTSIIS (amino acid residues 645-656 of HER2). This membrane-bound fragment (designated as ERBB2 p95 because of its molecular weight) shows potentially enhanced signalling activity. It has been speculated that the adverse prognosis observed in patients with high levels of ECD/ERBB2 may be related, at least in part, to an increase of truncated, signalling- competent, ERBB2 p95. [0265] Because the N-terminal membrane proximal sequence, EQRASPLTSIIS
(referred here as the "stumpy" region) potentially remains on cell surface after the proteolytic cleavage of the extracellular domain, the stumpy region is a potential target for therapeutic intervention. For example, Herceptin® (Trastuzumab), as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel, is indicated for the adjuvant treatment of patients with ERBB2-overexpressing, node-positive breast cancer. However,
Herceptin does not bind to the stumpy region of ERBB2. In contrast, an antibody that bind to the stumpy region of ERBB2 would be a more potent and effective inhibitor of the truncated, signalling-competent, ERBB2 p95.
Selection of scFvs that bind to the "stumpy" region by phage display [0266] Single chain fragment variable (scFv) moieties that bind to the membrane- proximal region of Her2 (ErbB2) that remains on the cell surface following cleavage and release of the soluble extra-cellular domain were identified following three rounds of selection using the Cambridge Antibody Technology (CAT) phage display libraries. Selection strategies are outlined in Figure 5. Three CAT libraries were used; the Bone Marrow Vaughan (BMV) library (Vaughan et al, 1996), the combined spleen (CS) library and the DP47 library (unpublished). The "stumpy peptide" and "scrambled peptide" (Figure 5A), each expressed with a biotin tag, were used during the selection and these peptides along with two Her2-Fc fusion proteins were used during the subsequent screening steps (see Table 3). The EKK sequence at the C termini of the stumpy peptide and scrambled peptide is predicted to maintain the helical structure predicted from the NMR (see, Goetz et al., 2001. Biochemistry 40: 6534-6540). For selections, aliquots of phage and magnetic streptavidin beads (Dynabeads M-280 streptavidin) were blocked separately in 3% milk/PBS for 1 hour at room temperature in a rotary mixer (20 rpm). Blocked phage were incubated with a 100 nM concentration of the scrambled de-selection peptide in round 1 (the amount of de-selection peptide decreased in subsequent rounds as the concentration of the selection peptide decreased), incubated at room temperature for 1 hour on a rotary shaker (20 rpm), mixed with blocked magnetic beads and incubated for a further hour. The de-selected library was collected by pelleting the beads using a magnetic separator. Biotinylated selection peptide (at various concentrations as indicated in Figure 5A) was incubated with the de-selected phage library for 2 hours at room temp on a rotary mixer (20 rpm) followed by a 15 minute incubation with pre-blocked magnetic beads. Beads were separated using a magnetic separator and washed 10 times with PBS/0.1% Tween 20 and 3 times with PBS. Bound phage were eluted by incubation with a 10 ug/ml solution of trypsin in PBS for 30 minutes at 37°C (100 rpm) followed by separation from the magnetic beads. [0267] Eluted phage were used to infect 10 ml of an E. coli TG 1 culture that had been grown to mid-logarithmic phase (corresponding to an OD6oo of ~ 0.5). Bacteria were infected with phage for 1 hour at 37°C with shaking at 150 rpm, concentrated following a centrifugation step and plated on 2X TY agar bioassay plates containing 2% glucose and 100 ug/ml ampicillin (2X TYAG). Various dilutions of E. coli culture infected with either input or output phage were also plated on 2X TYAG agar to determine phage titers. Following overnight growth at 300C1 10 ml of 2X TYAG medium was added to each bioassay plate and the cells were re-suspended by scraping the bacterial lawn. Glycerol was added to this cell suspension to give a final concentration of 17% and stored in aliquots at -8O0C until further use. In order to rescue phage for the next round of selection, 100 μl of this cell suspension was used to inoculate 20 ml 2X TYAG medium, which was grown at 37°C (300 rpm) to an OD600 of 0.3-0.5. Cells were then super-infected with 3.3 μl of MK13K07 helper phage and incubated at 37°C (150 rpm) for 1 hour. The cells were then centrifuged and the pellet re- suspended in a kanamycin/non-glucose containing medium (2X TY with 50 μg/ml kanamycin and 100 ug/ml ampicillin). This culture was grown overnight at 300C (300 rpm). Phage were harvested in the supernatant following centrifugation and were ready to use in the next round of selection as described above.
Example 12: ELISA to measure binding of scFvs expressed in the periplasm or purified to biotinylated Her2 protein constructs
[0268] A streptavidin-coated 96 well plate (Greiner) was washed three times with PBS/0.05% Tween 20 and blocked for 1 hour at room temperature in 3% skim milk/1% BSA/PBS. Plates were washed three times with PBS/0.05% prior to the addition of a 1 mg/ml solution of biotinylated Her2-Fc proteins (HerOOδP, HerO17P, HerO18P, HerO54P) or a biotinylated negative control murine lgG2a protein. Plates were incubated for one hour at room temperature. Peripreps were prepared as described in an earlier section and were blocked for 1 hour at room temperature in an equal volume of 6% skim milk/1% BSA/PBS. Blocked plates were washed five times with PBS/0.05% Tween 20 and 50 ml/well of blocked periprep (or purified scFv diluted in block buffer) were transferred to the appropriate plates and incubated for 1 hour at room temperature. A 1 ug/ml solution of Herceptin (in blocking buffer) was added to well H12 of each plate to serve as a positive control. Plates were washed five times with PBS/0.05% Tween 20 prior to the addition of a 1 :250 dilution of anti- myc peroxidase (Roche) or a 1 :5000 dilution of goat anti-human peroxidase (Southern Biotech) secondary antibody to detect bound scFv or Herceptin respectively. Plates were incubated for a further hour at room temperature and washed seven times with PBS/0.05% Tween 20. Signal was developed using TMB, the reaction stopped with H2SO4 and the absorbance read at 450 nm on an Envision plate reader (Perkin Elmer). Example 13: Conversion of scR, to IqG
[0269] Heavy and light chain V regions from scFv clones are amplified with clone- specific primers. PCR products are digested with appropriate restriction enzymes and subcloned into vectors containing human IgGI heavy chain constant domain (for VH domains) or vectors containing human lambda or kappa light chain constant domains as appropriate (VL domains). The closest human germlines of the VH and VL segments are determined and this information is used to indicate whether kappa or lambda light chain constant domains are used. Correct insertion of V region domains into plasmids is verified by sequencing of plasmid DNA from individual E. coli colonies. Plasmids are prepared from E. coli cultures by standard techniques and heavy and light chain constructs are co-transfected into COS cells using standard techniques. Secreted IgG is purified using protein A sepharose (Pharmacia) and buffer exchanged into PBS.
Example 14 Effect of SMIPS on HER2 Surface Expression and Ectodomain Shedding
[0270] To investigate the effect of SMIPs on Her2 surface expression and ectodomain shedding, 200,000 SKBR3 cells/well were plated in 24-well tissue culture plates and incubated overnight. The next day, the media was carefully removed and the cells were incubated for 24 hours with fresh media containing 10 ug/ml SMIPs (HerO67, HerO94,
Her102, Her116, HeM 46 or HerO18) or molar equivalent of antibody (HERCEPTIN® as a positive control, or Retuxan as a negative control). The table below shows the SMIPs and antibodies that were used. As additional controls, cells were also treated with either 1 nM pervanedate to increase ectodomain shedding, or 5ug/ml TIMP1 a protease inhibitor that results in blockage of Her2 cleavage.
Figure imgf000085_0001
[0271] The levels of shed Her2 ECD in the supernatant was determined by ELISA.
After 24 hours supernatants were harvested and the amount of shed Her2 ectodomain determined by ELISA, using HERCEPTIN® to capture shed ectodomain and anti-Her2 TA-1 antibody to detect the captured ectodomain. Cells were harvested using trypsin and cell surface Her2 was determined by flow cytometry by staining with the SMIP or antibody used for the treatment. Levels of Her2 were determined and compared to untreated cells stained with the same SMIP or antibody. [0272] HERCEPTIN® treated cells were not detected by ELISA. As shown in
Figures 5OA and 50B1 SMIPs decrease shedding of the Her2 ectodomain. As shown in
Figure 5OC and 5OD, anti-HER2 SMIPs of the invention decrease cell surface Her2.
[0273] Without being bound by theory, the mechanism for SMIPs' decreasing cell surface Her2 and shedding Her2 ectodomain may be that teh SMIP blocks Her2 cleavage, thus reducing shed ectodomain and production of p95 Her2. Alternatively, SMIPs could increase Her2 internalization, thus reducing cell surface ECD. Similar mechanisms have been described for HERCEPTIN®.
Example 15 Anti-HER2 SMIP Cross-blocking
[0274] To investigate the ability of Her2 SMIPs and antibodies to block each other's binding to cell surface Her2, cross-blocking was investigated using FMAT blocking buffer. SMIPs were labeled with FMAT Blue as per manufacturers directions (Applied Biosystems). Unlabeled competitor SMIPs or Antibodies were diluted to 40OnM in FMAT Blocking Buffer (44 ug/mL for SMIPs; 59.2 ug/mL for antibodies). Each protein was titrated 1 :3 in FMAt blocking buffer in duplicate V-bottom tissue culture 96-well plates in a final volume of 60ul/well. Cells (SKBR3) were added in 6OuI FMAT blocking buffer to give 36,000 cells/well. Plates were incubated for 1 hour at room temperature before adding FMAT Blue labeled antibodies at a concentration determined to give maximal staining in the absence of competing unlabeled SMIP or antibody (5 ug/mL for HERCEPTIN®; 2ug/ml_ for HER018, 10ug/ml_ for all other HER SMIPs1 and 2ug/ml for Rituxan and 2LM20-4 (anti-CD20 SMIP)). Plates were incubated at room temperateure for 45-60 minutes (10 minutes for Herceptin). Cells were spun down at 1250 rpm for 5 minutes and non-bound SMIPs and antibodies flicked off. Cells were resuspended in 12OuI FMAT Blocking Buffer and transferred to FMAT 96-well plates. Cells were allowed to settle for 5 minutes before being read on FL1 AB3200. The average of each duplicate value of FMAT staining was determined for each concentration of competing unlabeled protein.
[0275] Cross-blocking between Her2 SMIPs and antibodies is not necessarily indicated by epitope mapping. SMIPs and antibodies that bind different epitopes could nevertheless block each other due to binding stoichiometry and molecular size. Figure 51 shows a summary of the Her2 binding site possibilities for various SMIPs.
[0276] HERCEPTIN® binding is blocked by HERCEPTIN® and HER018 at low concentrations; HER067, HER102, HER146 at higher concentrations; and HER094 at very high concentrations. HER018 binding is blocked by HER018 and HERCEPTIN® at low concentrations; HER067, HER102, HER146 at higher concentrations; and HER094 at very high concentrations. HER067 binding is blocked by HERCEPTIN® and HER018 at low concentrations; HER067 and HER102 at higher concentrations; and HER094 and HER146 at very high concentrations. Also, HER067 binding is greatly enhanced by HER116 binding. [0277] HER094 binding is blocked by HERCEPTIN® and HER018 at low concentrations; HER067 and HER102 at higher concentrations; and HER094 and HER146 at very high concentrations. Also, HER094 binding is greatly enhanced by HER1 16 binding.
[0278] HER102 binding is blocked by HERCEPTIN® and HER018 at low concentrations; and HER146 and HER102 at higher concentrations. Also, HER102 binding may be slightly enhanced with HER1 16 binding.
[0279] HER1 16 binding is blocked by HER116 at low concentrations. No other SMIPs or antibodies blocked HER116 binding. HER146 binding is blocked by HERCEPTIN®, HER018 and HER102 at low concentrations; and HER146 at higher concentrations. Anti-CD20 Ab and SMIP binding is not blocked by any HER2 SMIPs or antibodies. [0280] The SMIP cross-blocking results are summarized in Figure 52.
[0281] Importantly, Her2 SMIPs that do not cross-block each other have the potential to simultaneously bind to a Her2 molecule. Accordingly, there may be an additive mechanism of action for Her2 binding with SMIPs and antibodies. Further, there is a possibility for a combination treatment with multiple SMIPs or with a combination of SMIP and antibody. SMIPs could also be potential partners for bispecific molecules such as Scorpions™ .
Example 16 Anti-HER2 SMIP Internalization Assay
[0282] We investigated the ability of anti-HER2SMIPS of the invention to internalize in various cell lines as follows.
Hum-ZAP Internalization Assay [0283] Hum-ZAP (Advanced targeting Systems) is a saporin-conjugated anti- human Ig that targets and eliminates cells using the internalization of an antibody or SMIP. Upon binding to a human IgG containing molecule, such as a SMIP or antibody, that recognizes an extracellular domain of a cell surface antigen, Hum-ZAP is taken inside the cell by antibody or SMIP-mediated internalization. The entrance of saporin into the cell will result in protein synthesis inhibition and eventual cell death after 2-4 days.
Cells in 90 μl of media (at a concentration of 2.5-5.0 x 103 cells/well) were added to 96-well plates and incubated overnight. The following day, cells were treated by either: a) the addition of 5 μl of a SMIP and media; b) the addition of 5 μl goat IgG-SAP (goat anti- human IgG negative control) and media; or c) the addition of 5 μl of a SMIP and Hum-ZAP (Saporin-conjugated goat anti-human IgG). Cells were incubated a further 96 hours before being assayed for proliferation using standard BrdU-incorporation and Hoechst nuclear staining. Internalization was observed as a reduction in cell proliferation an plotted as percent of untreated control.
[0284] We observed that, of the SMIPs tested, HER116 was best internalized by cells - these results agree with the fluorescence internalization experiments with "stumpy" binders.
[0285] Stumpy binders (HER156 and HER169) were observed to internalize in MDA-MB-361 JL, MDA-MB-453, and BT-474 cells and, to some degree, in SKBR3 cells. In addition, all SMIPs were internalized to some degree in BT474 and SKBR3 cells. [0286] No detectable SMIP internalization, however, was observed in JIMT-1 and
MDA-MB-361 (ATCC) cells. This may be due to the fact that MDA-MB-361 (ATCC) cells grow slowly. Thus, longer treatment times with increased cell numbers may be necessary in order to detect a response.
Fluorescence Assay [0287] MDA-MB-361 cells were grown in 96-well plate format and treated with anti-
Her2 SMIPs1 Herceptin (Here) or control anti-CD20 SMIP for indicated times. Media was removed and cells fixed (4% paraformaldehyde) and permeabilized (0.2% TritonlOO). Cell surface or intracellular SMIPs or monoclonal antibodies were detected by staining with FITC- labeled anti-hulgG-Fc (see Figure 53A-F, panels A and B or with rabbit anti-Her2 mAB (Cell Signaling Technologies) with secondary FITC-labeled Goat-anti-Rabbit IgG (Molecular
Probes) (panel C) . Fluorescent image detection was visualized by Cellomics High Content assay.
[0288] Her116 demonstrated rapid binding and internalization of SMIP (Panel A:10min; Panel B: 1hr) and cell surface Her2 (Panel C: 1 hr) similar to Herceptin mAB. In contrast, Her146 treatment demonstrated slower kinetics of cell surface binding that was sustained for longer time periods (Panel B: 1hr) and confirmed with anti-Her2 cell surface localization (Panel C: 1hr). Control anti-CD20 SMIP did not display binding at any time point as anticipated.
CVPHER5E Assay
[0289] SMIPs and antibodies were labeled with CypHerδE (GE Healthcare) as per manufacturers direction. CypHerδE has little or no fluorescence at physiological pH, but fluoresces at low pH (e.g., when internalized into lysosomal compartments). Cells were plated in serum-free media and placed on ice for 5-10 minutes. Cells were then washed (1x) with cold media containing 1% FBS. Dilutions of CypHerδE labeled SMIPs or antibodies in ice cold serum-free media were added to cells and incubated on ice for 45 minutes. Cells were washed (1x) with ice cold media containing 1% FBS. Room temperature media containing 1% FBS (without phenol red, pH 7.6) and Hoechst nuclear stain (Invitrogen) were added to cells and the . Cells were then imaged using an InCeII analysis System (GE Healthcare) using the 2OX objective at 10 or 30 minute intervals for 2-4 hours. Cells were then fixed with formaldehyde, permeabilized, blocked and stained with an anti-human Alex- 488 secondary mAb (anti-human IgG H+L, Invitrogen) and re-imaged on the InCeII to verify SMIP or antibody binding. CypHerδE is imaged in the red channel (650-700nm), Hoechst in the blue channel (387-525nm) and the Alex-488 secondary antibody in the green channel (485-525nm). [0290] HER018 and HER116 were rapidly internalized- within 10 minutes. We confirmed the presence of SMIP binding after fixation with an anti-human Fc secondary Ab. The presence of SMIP binding was confirmed after fixation with an anti-human Fc secondary Ab. We found that HER067, HER146, HER156, and HER169 were internalized more slowly. We observed some internalization of these SMIPs by 4 hours. We found that Herceptin was internalized at a faster rate in SMIP format (HER018) than as Ab. In general, we found that "stumpy binders" were internalized over a longer period of time. Some had internalization by 4 hours. Without wishing to be bound by any theory, we believed that they could induce cell death over a period of days through internalizing of a co-incubated toxin -conjugated anti human secondary antibody.
Example 17 Effector Functions of Anti-HER2 SMIPS
[0291] SKBR3 cells were harvested with trypsin and washed. Cells were labeled with BADTA (Perkin Elmer) by incubating 2x106 cells in 2ml media with 20μl BADTA mix (5μl BADTA reagent, 2μl PF127, 13μl DMSO) for 20 minutes at 37C. Labeled cells were washed with PBS (4x) and resuspended in media at 400,000 cells/ml. Cells (20,000 cells in 50μl) were aliquoted into a V-bottom plate and 50μl of 2x SMIP or antibody were added. To a 50μl aliquot of loaded target cells, 150 μl media were added and immediately spun down; background spontaneous release counts were determined in the 100μl supernatant. To a second 50μl aliquot, 150 μl 2% NP40 in media were added to lyse cells in order to determine maximum lysis. Effector cells (PBMC: peripheral blood mononuclear cells) were added to samples and incubated for 2 hours at 37C. Plates were centrifuged to pellet cells/debris. Cleared supernatant (20μl) was transferred to a flat bottom plate to which 200μl of Europium solution was added and incubated for 15 minutes at room temperature while shaking. Fluorescence was measured (excitation 335nm, emission 615nm, cutoff 590nm, delay 250μs, and integration 1250μs).
[0292] As shown in Figure 54, HER116m HER033/067 and HER094 have good to moderate FcDCC activity that is comparable to that of HERCEPTIN® and HER018. In contrast, we observed no CDC activity with anti-HER2 SMIPS1 HERCEPTIN® or HLA ABC in SKBR3 cells (see Figure 55) or MDS-MB-361-JL cells (data not shown). Example 18 Serum Stability of Anti-HER2 SMIPS
[0293] Stability of SMIPs in mouse plasma was determined by incubating SMIPs (200ug/ml) in mouse plasma or PBS at 37C or 4C for up to 96 hours, with samples removed at intermediate times. A dilution series was made for each SMIP sample and the concentration was determined by ELISA using plates coated with a Her2 ECD murine Fc fusion protein (Her2SIIS::muFc). Captured SMIP was detected using a HRP-conjugated secondary anti mouse Fc secondary antibody. Mouse plasma alone or an anti-CD20 antibody, were used as negative controls in these experiments.
[0294] Results are shown in Figure 56A. The effects of different incubation times and different temperatures had little effect on the binding of HER067 or HER 146 with Her2- SIIS at all of the concentrations tested. These results suggest that HER067 and HER146 are stable in plasma at physiologically-relevant temperatures and for extended periods of time. Repeat experiments that compared samples incubated in plasma with those that were incubated in PBS for 72 hr provided results that agreed with the first assay shown in (see Figure 56B).
Example 19 Determination of Mass of Soluble HER2 Receptor-Binding Molecule Complex [0295] In order to determine the binding ratios of SMIPs to Her2 receptors, the mass of Her2 receptor/binding domain complexes was analyzed. Figures 57A and 57B show predicted masses of various SMIP/receptor complexes. SMIP (or mAb) was mixed with a soluble Her2 receptor at a 3:1 Molar ratio (an alternate mixture with a SMIP:receptor Molar ratio of 1 :3 was also used), and the mixture was incubated at room temperature overnight. The mixture volumes were then normalized to 110 μl, and 100 μl of the
SMIP/receptor mixture were subjected to size-exclusion chromatography combined with refractive index, multiple angle laser light scattering (SEC-RI-MALLS), using a TOSOH TSK G4000 SWχL column. The mass of the resolved peaks was analyzed using ASTRA software (Wyatt Technology Corporation, CA). The results of the mass analysis are shown in Figure 58.
[0296] Our observations suggest that each Herceptin, HER018, and HER095 molecule binds two monomeric soluble Her2 receptors simultaneously. This binding appeared to be complete with 2 Her2 molecules per SMIP when the receptor was in 3x excess. In contrast, it appears that HER067 and HER146 can only bind one monomeric soluble Her2 receptor. This binding appeared to be incomplete/poor with 0 to 1 Her2 molecules per SMIP when the receptor was in 3x excess. The data also support that two Herceptin, HER018 or HER095 molecules can bind to each dimeric soluble Her2 receptor, when the SMIP was in 3x excess. Alternatively, it appears that only one HER067 or HER146 molecule binds each dimeric soluble Her2 receptor, even when the SMIP was in 3x excess.
Example 20 Drug Combination Studies
[0297] Cell cycle data suggested that MDA-MB-453 and MDA-MB-361_JL cells treated with Her146 and other SMIPs were driven into and through S-phase before being arrested in G1. The ability for SMIPs to sensitize cells to chemotherapeutic agents was determined by performing standard proliferation assays (BrdU incorporation and/or Hoechst nuclear staining) on cells treated with SMIP/antibody prior to treatment with chemotherapeutic agents (Cisplatin, Taxol, Doxorubicin or Gemcitabine). Cells (2500-5000 cells per 96 well) were treated with SMIP for 24 or 72 hours prior to the addition of chemotherapy. Cells were treated with the combination of SMIP/antibody and therapeutic an additional 24 hours before the cells were quantitated by counting cells using the nuclear stain, Hoechst, or by the ability of live cells to incorporate BrdU using standard assays. A 5- fold dilution series was run for each assay/treatment with a maximal concentration of SMIP of 182nM and 10OuM Cisplatin, 10OnM Taxol, 100OnM Doxorubicin, or 10OnM Gemcitabine with the ratio remaining constant for each dilution. The combination of SMIP and chemotherapy was compared to either SMIP or chemotherapy alone. Dose response curves of cells pre-treated with HER146 and then treated with various chemotherapeutic agents or combinations thereof are shown in Figure 59A-D.
[0298] We found that Her2 SMIPs could have additive effects when administered with chemotherapeutic agents. For example, MDA-MB-453 cells treated with HER146 were more sensitive to chemotherapeutic agents (e.g., Cisplatin, Taxol, and Doxorubicin). MDA- MB-361-JL cells treated with Her146 were more sensitive to some chemotherapeutic agents (e.g., Cisplatin, Taxol, and Gemcitabine) but not others (e.g., Doxorubicin).
Example 21: Assessment of Target Binding Specificity
[0299] We investigated the target-binding specificity of the anti-HER2 SMIPS referred to herein as "stumpy" binders by immunoprecipitation. Cells was solubilized in either Radio lmmuno Precipitation Assay (RIPA) lysate buffer or Nonidet P-40 lysis buffer (a more gentle detergent than those in RIPA buffer) containing a cocktail of protease inhibitors. Cleared lysate protein concentrations were determined using a standard protein assay (e.g. Bradford assay). Her2 was immunoprecipitated from 1mg RIPA lysate or 2mg Nonidet P-40 lysate using 5ug of SMIPs1 5ug human IgG (as negative control) or 2ug mouse monoclonal antibody, 3B5, against the intracellular region of Her2 (positive control), lmmunoprecipitated protein is pulled down with protein A or protein G beads, washed and separated by SDS- PAGE. Separated proteins were transferred to membrane by standard Western blotting and proteins detected with a primary rabbit polyclonal antibody against an intracellular domain of Her2 (amino acids 975-1025; Bethyl #A300-621A) and a IRDye labeled donkey anti-rabbit secondary (LI_COR #926-32223) and visualized using a LI-COR infrared fluorescence labeling and detection system. The secondary antibody we used was 3 x 1 :5000 IRDye 680 Donkey anti-rabbit lgG(H+L) (LI-COR #926-32223, lot B70215-02). The results are presented in Figure 60.
[0300] Alternatively, we solubilized Ramos, JIMT-1 and MDA-MB-361 ATCC cells with Nonidet P-40 lysis buffer (a gentler detergent than those in RIPA buffer), and immunoprecipitated HER2 from 2 mg of the lysate using 5 μg Human IgG as negative control, 5 μg SMIPs (HeM 16, Her156, and Her 169), and 2 μg mouse monoclonal 3B5 as positive control, respectively. We ran the immunoprecipitates on polyacrylamide gels and performed Western blotting experiment. The primary antibody we used was 3 x 1 :200 Rabbit polyclonal to ErbB2 C-term (abeam #ab2428-1 , lot 212287). The secondary antibody we used was 3 x 1 :5000 IRDye 800CW Donkey anti-Rabbit lgG(H+L) (LI-COR #926-32213, lot B70416-01 ). The results are presented in Figure 61. [0301] We found that HER156 and HER169 are capable of binding full-length
HER2. It was unclear to us whether HER156 and HER169 could bind Her2 p95 ("Stumpy;" cleaved ErbB2 that should run at 95 KDa). For example, it was not clear to us whether p95 can be immunoprecipitated at detectable levels from SKBGR3 cells by either HER156 or HER169. It was possible that there was too little p95 in SKBR3 cells for detection. We also observed that the immunoprecipitation pattern of HER116, which binds to L1/CR1 domains of Her2 not present on p95, looked the same as for 3B5, which binds intracellular domain of Her2 found on full length Her2 and p95 "stumpy." This was possibly due to sample degradation observed post immunoprecipitation.
[0302] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supercede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.
[0303] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the application, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
[0304] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.
Sequence Table
Her2_S1R2A_CS_1F7
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGT NYAQKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCARDSTMAPGAFDIWGRGTLVTV SS
(SEQ ID NO:1 )
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSVAPGQTARMTCGGNNIESKTVHWYQQKPGQAPVLWYNDNVRPSGIPAR FSGSNSGNTATLTINRVEAGDEADYYCQVWDSSRDQGVFGGGTKLTVLGA (SEQ ID NO:2)
Her2_S1 R2A_CS_1 D11
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGSEVRRPGSSVRVSCTASGDTSSSFTVNWLRQAPGQGLEWMGGITPMFGTAN YAQMFEDRVTITADEMELSGLTSEDTAVYFCATGPSDYVWGSYRFLDTWGRGTTVTVSS (SEQ ID NO:3)
VL with CDR1 , CDR2 and CDR3 underlined
QAVLTQPSSVSAAPGQEVSISCSGARSNVGGNYVSWYQHLPGTAPKLLIYDNNKRPSGMP DRFSGSKSGTSATLGITGVQTEDEADYYCATWDSSLSAWFGGGTKLTVLGA (SEQ ID NO:4)
Her2_S1R2C_CS_1D3
VH with CDR1 , CDR2 and CDR3 underlined
QVQLVQSGSEVRRPGSSVRISCTASGDTSSSFTVNWVRQAPGQGLEWMGGITPMFGTAN YAQVFEDRVTIIADEMELSGLTSEDTAVYFCATGPSDYVWGSYRFLDRWGRGTLVTVSS (SEQ ID NO:5)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSAAPGQKVTISCSGGRSSIGNNYVSWYQHLPGTAPKLLIYDNNQRPSGIPD RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWFGGGTKVTVLGA (SEQ ID NO:6)
Her2_S1 R2C_CS_1 H12
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVETGGGLVQPGGSLRLSCAASGFTFSSYGMNWVRQAPGKGLEWVSYISSSGNTIFY ADSVKGRFTISRDSAKNSVSLQMNSLRDEDTAVYYCASYYSYYYGMDAWGQGTMVTV (SEQ ID NO:7) VL with CDR1 , CDR2 and CDR3 underlined
SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDYSLSGWVFGGGTKVTVLGA (SEQ ID NO:8)
Her2_S1 R2A_CS_1 D3
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGAEVKKPGASVKVSCKASGYSFTAFYIHWVRQAPGQGLEYLGWIDPNTGATKY AQRFQGRVIMTWDTSITTATMELSRLTSDDSAVYYCVRDLREWGYELSVEYWGRGTLVTV SS
(SEQ ID NO:9)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGA (SEQ ID NO:10)
Her2_S1 R3B2_BMV_1 E1
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVETGGGWQPGGSLSLSCAASGFTFSSYGMQWVRQAPGKGLEWVAFIRYDGSSEY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGRTLESSLWGKGTLVTVSS (SEQ ID NO:11 )
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVP DRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTRTKLTVLGA (SEQ ID NO:12)
Her2_S1 R3C1_CS_1D3
VH with CDR1, CDR2 and CDR3 underlined
QVQLQESGPGLVKPSQTLSLTCGISGDSVSSNSAAWNWIRQSPTRGLEWLGRTYYRSSW YHNYAPSMNSRLTIIADTSKNQFSLQLNSVTPEDTAVYYCASGWAFDVWGRGTLVTVSS (SEQ ID NO:13)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGSPGQSVTISCTGTSSDVGAYDFVSWYQQHPGKAPKLMIYEVNKRPSGV PDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSKNLLFGGGTKLTVLGA (SEQ ID NO:14) Her2_S1 R3B2_DP47_1 E8
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQSGADWYFDLWGRGTLVTVSS (SEQ ID NO:15)
VL with CDR1 , CDR2 and CDR3 underlined
QAVLTQPSAVSGAPGQRVTISCTGTSSNIGTNYLVHWYQQRPGTAPQLLVSGNNTRPSGV TDRFSVSKSATSASLAITGLQAEDEADYYCQTYDINLRVWVFGGGTKVTVLGA (SEQ ID NO:16)
Her2_S1 R3B2_BM V_1 G2
VH with CDR1 , CDR2 and CDR3 underlined
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTN YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVPGVSGSYPDYYYMDVWGKG TLVTVSS
(SEQ ID NO:17)
VL with CDR1 , CDR2 and CDR3 underlined
DIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRF SGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKRA (SEQ ID NO:18)
Her2_S1 R3B2_BMV_1 H5
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGGGLVRPGGSLRLSCAASGFSFSDYYMTWIRQIPGKGLEWVAVIWNDGSDRYY ADSVKGRFTISRDNSKNTLFLQMSSLRDEDTALYYCVRGGPTASSGFDYWGRGTLVTVSS (SEQ ID NO:19)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYLQHPGKAPKLMIYEGSKRPSGVS NRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVLGA (SEQ ID NO:20)
Her2_S1 R3C1_CS_1 A6
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGAEVKKPGESLKISCKGFGYNFRSAWIGWVRQMPGKGLEWMGVIYPGDSDVR YSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRPVGQWVDSDYWGKGTLVTVSS (SEQ ID NO:21) VL with CDR1, CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNTVNWYQQLPGTAPKLLIYTSNQRPSGVPA RFSASNSGTSASLAISGLRSEDEADYYCAAWDDKLSGAVFGGGTKLTVLGA (SEQ ID NO:22)
Her2_S1 R3B2_DP47_1 C9
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWRPLLDYHFDQWGQGTMVTV SS
(SEQ ID NO:23)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQTVTISCSGSSSNIGSSWNWYQQFPGTAPKVLVYSNTQRPSGVP DRFSGSRSGTSASLAISGLQSEDEADYYCLAWDASLNGWVFGGGTKLTVLGA (SEQ ID NO:24)
Her2_S1 R3B2_DP47_1 E10
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSGYDDPDSWGRGTTVTVSS (SEQ ID NO:25)
VL with CDR1 , CDR2 and CDR3 underlined
HVILTQPPSTSGTPGQTVTISCSGSSSNIGSHYVYWYQQLPGTAPKLLIYRNNQRPSGVPD RFSGSKSGTSASLAISGLRSEDETDYYCAAWDDSLSGRVFGTGTKLTVLGA (SEQ ID NO:26)
Her2_S1 R3C1_CS_1 B10
VH with CDR1 , CDR2 and CDR3 underlined
QVQLQQSGAEVKKPGSSVKVSCKASGGTISNYAISWVRLAPGQGLEWMGSIVPLHGTTNF AQKFQGRVTITADESTSTSYMEVNVLTYEDTAMYYCASLNWGYWGRGTLVTVSS (SEQ ID NO:27)
VL with CDR1 , CDR2 and CDR3 underlined
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPDSAPTTVIYEDNRRSSGVPD RFSGSIDSNSASLSISGLKTEDEADYYCQSYDSSGHWFGGGTKLTVLGA (SEQ ID NO:28) Her2_S1 R3A1_BMV_1 F3
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVESGEGLVKPGGSLRLSCTASGFTFRSYSLNWVRQAPGQGLEWVSSISSTSTYIYYA DSVKGRFTISRDDAKNTLYLQMNSLRAEDTAAYYCVRLGSGGGYFPDYWGRGTLVTVSS (SEQ ID NO:29)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVLGA (SEQ ID NO:30)
Her2_S1R3B1_BMV_1G11
VH with CDR1 , CDR2 and CDR3 underlined
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSTYAMSWARQAPGKGLEWVSSISGDGGRIL DADSAKGRFTISRDNSKNTLYLQMNGLRVEDTALYYCARADGNYWGRGTMVTVSS (SEQ ID NO:31)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVLGA (SEQ ID NO:32)
Her2_S1 R3A1_BMV_1G4
' VH with CDR1 , CDR2 and CDR3 underlined
QVQLVESGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQRLEWMGWINAGNGNTK YSQKFQGRVTITRDTSASTAYMELRSLRSDDTAVYYCARGRSYGHPYYFDYWGQGTLVTV SS
(SEQ ID NO:33)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVLGA (SEQ ID NO:34)
Her2_S1 R3B1_BMV_1 H11
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAGIFYDGGNKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYYYMDVWGKGTTVTVSS (SEQ ID NO:35) VL with CDR1, CDR2 and CDR3 underlined
QSVLTQPPSVSGAPGQRVTISCTGRSSNIGAGHDVHWYQQLPGTAPKLLIYGDSNRPSGV PDRFSGSRSGTSASLAITGLQAEDEADYYCQSYDSSLRGSVFGGGTKVTVLGA (SEQ ID NO:36)
Her2_S1R3A1_CS_1B9
VH with CDR1 , CDR2 and CDR3 underlined
KVQLVQSGTEVKKPGESLKISCQGSGYRFSSDWIAWVRQMPGKGLEWMGIVYPGDSDTR
YSPSFQGQVTISADKSISTAYLQWSGLKASDTAKYYCARVQQAVGAKGYAMDVWGKGTLV
TVSS
(SEQ ID NO:37)
VL with CDR1 , CDR2 and CDR3 underlined
QTWIQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYRQTPGQAPHTLIHNTKIRSSGVP DRFSGSILGNNAALTITGAQADDESDYYCLLYMGSGIYVFGGGTKLTVLGA (SEQ ID NO:38)
Her2_S1 R3B1_BMV_1 H9
VH with CDR1 , CDR2 and CDR3 underlined
QVQLQESGAGLVKPSGTLSLTCAVSGGSISSGNWWSWVRQPPGKGLEWIGEISHSGSTN YNPSLKSRVTISVDKSKNQFSLNLSSVTAADTAVYYCARVRGTVGDTRGPDYWGQGTLVT VSS
(SEQ ID NO:39)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVLGA (SEQ ID NO:40)
Her2_S1 R3A1_CS_1 B10
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGAEVKKPGASVRVSCKGSGNTFTGHYIHWVRQAPGQGLEWLGWIDPNTGDIQ YSENFKGSVTLTRDPSINSVFMDLIRLTSDDTAMYYCAREGAGLANYYYYGLDVWGRGTM VTVSS
(SEQ ID NO:41 )
VL with CDR1 , CDR2 and CDR3 underlined
QTWLQEPSFSVSPGGTVTLTCGLNFGSVSTAYYPSWYQQTPGQAPRTLIYGTNIRSSGVP DRFSGSIVGNKAALTITGAQTEDESDYYCALYMGSGMLFGGGTKVTVLGA (SEQ ID NO:42) Her2_S1R3B1_BMV_1C12
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSIKY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGEYSGYDTSGYSNWGQGTLV
TVSS
(SEQ ID NO:43)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQRLPGAAPQLLIYNNDQRPSGIPD RFSGSKSGTSGSLVISGLQSEDEADYYCASWDDSLNGRVFGGGTKLTVLG (SEQ ID NO:44)
Her2_S1 R3C1_BMV_1 H11
VH with CDR1 , CDR2 and CDR3 underlined
GVQLVESGGGLVKPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSAISGSGGSTY YADSVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDTSGWYGDGMDVWGRGTLVT VSS
(SEQ ID NO:45)
VL with CDR1 , CDR2 and CDR3 underlined
DIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRF SGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKRA (SEQ ID NO:46)
Her2_S1R3B1_BMV_1A10
VH with CDR1 , CDR2 and CDR3 underlined
QMQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSIKY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTGVYYCSKDRYSSGWYSSDAFDIWGRGTM VTVSS
(SEQ ID NO:47)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCHSRDSSGNHVLFGGGTKLTVLGA (SEQ ID NO:48) Her2_S1 R3A1_CS_1 D11
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNHWIAWVRQMPGKGLEWMGIIYPGDSETRY SPSFQGHVTISADKSISTAYLQWSTLKDSDSAMYFCVRQARGWDDGRAGYYYSGMDAWG QGTLVTVSS
(SEQ ID NO:49)
VL with CDR1 , CDR2 and CDR3 underlined
QAWLQEPSFSVSPGGTVTLTCGLRSGSVSTSHYPSWYQQTPGQAPRTLIYSTNTRSSGV PDRFSGSILGNKAALTITGAQADDESNYYCMLYMGSGMYVFGGGTKVTVLGA (SEQ ID NO:50)
Her2_S1 R3C1_DP47_1 H1
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSGSHFPFFDSWGQGTMVTVS S
(SEQ ID NO:51 )
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSVAPGQTARITCGGDKIGHKSVHWYQQKPGQAPVLLVYDDRKRPSGIPER FSGSNSGNTATLTISRVEAGDEAAYHCQVWDRSSDPYVFGTGTKVTVLGA (SEQ ID NO:52)
Her2_S1 R3A1_CS_1 B12
VH with CDR1 , CDR2 and CDR3 underlined
QVQLVQSGAEVKKPGASVKVSCQASGYTFSGHYMHLVRQAPGQGLEWMGWIHPTSGGT TYAQKFQGRWMTRDTSISTAYMELSRLTSDDTAVYYCARMSQNYDAFDIWGQGTMVTVS S
(SEQ ID NO:53)
VL with CDR1 , CDR2 and CDR3 underlined
QAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYDVNWYQQFPGTAPKIIVYGDRPSGAPDR FSGSKSGTSASLAITGLRAEDEADYYCQSWDSRLSSYVFGTGTKVTVLGA (SEQ ID NO:54) Her2_S1 R3B1_BMV_1 H5
VH with CDR1 , CDR2 and CDR3 underlined
QVQLQESGGGWQPGGSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVASVRNDGSNT
YYTDSVKDRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAKSRRVMYGTSYYFDYWGRGTL
VTVSS
(SEQ ID NO:55)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVLGA (SEQ ID NO:56)
Her2_S1 R3A1_DP47_1 A6
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGIDPLWSGYYTPLDYWGRG TMVTVSS
(SEQ ID NO:57)
VL with CDR1 , CDR2 and CDR3 underlined
HVILTQPPSASGTPGQRVTISCSGSSSNIGSNSVSWYQQLPGTAPKLLMYTNNQRPSGVPD RFSGSKSGTSASLAISGLQSEDEADYYCATWDASLNTWVFGGGTKVTVLGA (SEQ ID NO:58)
Her2_S1 R3B1_DP47_1 E1
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSGSDYWGQGTMVTVSS (SEQ ID NO:59)
VL with CDR1 , CDR2 and CDR3 underlined
NFMLTQPHSVSGSPGKTVTISCTRSSGYIDSKYVQWYQQRPGSAPTTVIYEDNRRPSGVP DRFSGSIDSNSASLTISGLETEDEADYYCQSYDDTNWFGGGTKVTVLGA (SEQ ID NO:60) Her2_S1R3B1_BMV_1A1
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVQSGAEVKEPGASVKVSCKASGYDFSNYGFSWVRQAPGQGLEWMGWISSYNGYT NYAQRLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDRGLGNWYFDLWGQGTLVT VSS (SEQ ID NO:61)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVLGA (SEQ ID NO:62)
Her2_S1R2A_CS_1F7
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSVAPGQTARMTCGGNNIESKTVHWYQQKPGQAPVLWYNDNVRPSGIPAR FSGSNSGNTATLTINRVEAGDEADYYCQVWDSSRDQGVFGGGTKLTVL (SEQ ID
NO: 63)
Her2_S1R2A_CS_1D11
VL with CDR1 , CDR2 and CDR3 underlined
QAVLTQPSSVSAAPGQEVSISCSGARSNVGGNYVSWYQHLPGTAPKLLIYDNNKRPSGMP DRFSGSKSGTSATLGITGVQTEDEADYYCATWDSSLSAWFGGGTKLTVL (SEQ ID NO: 64)
Her2_S1R2C_CS_1D3
VH with CDR1 , CDR2 and CDR3 underlined
QVQLVQSGSEVRRPGSSVRISCTASGDTSSSFTVNWVRQAPGQGLEWMGGITPMFGTAN YAQVFEDRVTIIADEMELSGLTSEDTAVYFCATGPSDYVWGSYRFLDNWGRGTLVTVSS (SEQ ID NO: 65)
Her2_S1R2C_CS_1D3
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSAAPGQKVTISCSGGRSSIGNNYVSWYQHLPGTAPKLLIYDNNQRPSGIPD RFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWFGGGTKVTVL (SEQ ID
NO: 66)
Her2_S1 R2C_CS_1 H 12
VH with CDR1 , CDR2 and CDR3 underlined
EVQLVETGGGLVQPGGSLRLSCAASGFTFSSYGMNWVRQAPGKGLEWVSYISSSGNTIFY ADSVKGRFTISRDSAKNSVSLQMNSLRDEDTAVYYCASYYSYYYGMDAWGQGTMVTVSS (SEQ ID NO: 67) Her2_S1 R2C_CS_1 H12
VL with CDR1, CDR2 and CDR3 underlined
SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDYSLSGWVFGGGTKVTVL (SEQ ID
NO: 68)
Her2_S1R2A_CS_1D3
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL (SEQ ID NO: 69)
Her2_S1 R3B2_BMV_1 E1
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVP DRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTRTKLTVL (SEQ ID
NO: 70)
Her2_S1 R3C1_CS_1 D3
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGSPGQSVTISCTGTSSDVGAYDFVSWYQQHPGKAPKLMIYEVNKRPSGV PDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSKNLLFGGGTKLTVL (SEQ ID NO: 71 )
Her2_S1 R3B2_DP47_1 E8
VL with CDR1 , CDR2 and CDR3 underlined
QAVLTQPSAVSGAPGQRVTISCTGTSSNIGTNYLVHWYQQRPGTAPQLLVSGNNTRPSGV TDRFSVSKSATSASLAITGLQAEDEADYYCQTYDINLRVWVFGGGTKVTVL (SEQ ID NO: 72)
Her2_S1 R3B2_BM V_1 G2
VL with CDR1 , CDR2 and CDR3 underlined
DIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRF SGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK (SEQ ID NO: 73)
Her2_S1 R3B2_BMV_1 H5
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYLQHPGKAPKLMIYEGSKRPSGVS NRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVL(SEQ ID NO: 74) Her2_S1R3C1_CS_1A6
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNTVNWYQQLPGTAPKLLIYTSNQRPSGVPA RFSASNSGTSASLAISGLRSEDEADYYCAAWDDKLSGAVFGGGTKLTVL (SEQ ID
NO: 75)
Her2_S1 R3B2_DP47_1 C9
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQTVTISCSGSSSNIGSSWNWYQQFPGTAPKVLVYSNTQRPSGVP DRFSGSRSGTSASLAISGLQSEDEADYYCLAWDASLNGWVFGGGTKLTVL (SEQ ID NO: 76)
Her2_S1 R3B2_DP47_1 E10
VL with CDR1 , CDR2 and CDR3 underlined
HVILTQPPSTSGTPGQTVTISCSGSSSNIGSHYVYWYQQLPGTAPKLLIYRNNQRPSGVPD RFSGSKSGTSASLAISGLRSEDETDYYCAAWDDSLSGRVFGTGTKLTVL (SEQ ID NO: 77)
Her2_S1R3C1_CS_1B10
VL with CDR1 , CDR2 and CDR3 underlined
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPDSAPTTVIYEDNRRSSGVPD RFSGSIDSNSASLSISGLKTEDEADYYCQSYDSSGHWFGGGTKLTVL (SEQ ID NO: 78)
Her2_S1 R3A1_BMV_1 F3
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVL (SEQ ID NO: 79)
Her2_S1R3B1_BMV_1G11
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVL (SEQ ID NO: 80)
Her2_S1 R3A1_BMV_1 G4
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVL (SEQ ID NO: 81 ) Her2_S1 R3B1_BMV_1 H11
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSGAPGQRVTISCTGRSSNIGAGHDVHWYQQLPGTAPKLLIYGDSNRPSGV PDRFSGSRSGTSASLAITGLQAEDEADYYCQSYDSSLRGSVFGGGTKVTVL (SEQ ID NO: 82)
Her2_S1R3A1_CS_1B9
VL with CDR1 , CDR2 and CDR3 underlined
QTWIQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYRQTPGQAPHTLIHNTKIRSSGVP DRFSGSILGNNAALTITGAQADDESDYYCLLYMGSGIYVFGGGTKLTVL (SEQ ID NO: 83)
Her2_S1 R3B1_BMV_1 H9
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVL (SEQ ID NO: 84)
Her2_S1 R3A1_CS_1 B10
VL with CDR1 , CDR2 and CDR3 underlined
QTWLQEPSFSVSPGGTVTLTCGLNFGSVSTAYYPSWYQQTPGQAPRTLIYGTNIRSSGVP DRFSGSIVGNKAALTITGAQTEDESDYYCALYMGSGMLFGGGTKVTVL (SEQ ID NO: 85)
Her2_S1R3B1_BMV_1C12
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQRLPGAAPQLLIYNNDQRPSGIPD RFSGSKSGTSGSLVISGLQSEDEADYYCASWDDSLNGRVFGGGTKLTVL (SEQ ID NO: 86)
Her2_S1 R3C1_BMV_1 H11
VL with CDR1 , CDR2 and CDR3 underlined
DIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRF SGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK (SEQ ID NO: 87
Her2_S1R3B1_BMV_1A10
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCHSRDSSGNHVLFGGGTKLTVL (SEQ ID NO: 88) Her2_S1 R3A1_CS_1 D11
VL with CDR1, CDR2 and CDR3 underlined
QAWLQEPSFSVSPGGTVTLTCGLRSGSVSTSHYPSWYQQTPGQAPRTLIYSTNTRSSGV PDRFSGSILGNKAALTITGAQADDESNYYCMLYMGSGMYVFGGGTKVTVL (SEQ ID NO: 89)
Her2_S1 R3C1_DP47_1 H1
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSVAPGQTARITCGGDKIGHKSVHWYQQKPGQAPVLLVYDDRKRPSGIPER FSGSNSGNTATLTISRVEAGDEAAYHCQVWDRSSDPYVFGTGTKVTVL (SEQ ID NO: 90)
Her2_S1R3A1_CS_1B12
VL with CDR1 , CDR2 and CDR3 underlined
QAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYDVNWYQQFPGTAPKIIVYGDRPSGAPDR FSGSKSGTSASLAITGLRAEDEADYYCQSWDSRLSSYVFGTGTKVTVL (SEQ ID NO: 91)
Her2_S1 R3B1_BMV_1 H5
VL with CDR1 , CDR2 and CDR3 underlined
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRF SGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWFGGGTKLTVL (SEQ ID NO: 92)
Her2_S1 R3A1_DP47_1 A6
VL with CDR1 , CDR2 and CDR3 underlined
HVILTQPPSASGTPGQRVTISCSGSSSNIGSNSVSWYQQLPGTAPKLLMYTNNQRPSGVPD RFSGSKSGTSASLAISGLQSEDEADYYCATWDASLNTWVFGGGTKVTVL (SEQ ID NO: 93)
Her2_S1 R3B1_DP47_1 E1
VL with CDR1 , CDR2 and CDR3 underlined
NFMLTQPHSVSGSPGKTVTISCTRSSGYIDSKYVQWYQQRPGSAPTTVIYEDNRRPSGVP DRFSGSIDSNSASLTISGLETEDEADYYCQSYDDTNWFGGGTKVTVL (SEQ ID NO: 94)
Her2_S1 R3B1_BMV_1 A1
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGV SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVL (SEQ ID NO 95) Her2_S1R2A_CS_1F7
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG
GTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGAC
AGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCA
CAAACTATGCACAGAAGTTTCAGGGCTGGGTCACCATGACCAGGGACACGTCCATCAG
CACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGT
GCGAGAGATTCTACTATGGCCCCAGGTGCTTTTGATATCTGGGGCCGAGGCACCCTGG
TCACCGTCTCGAGT (SEQ ID NO: 96)
Her2_S1R2A_CS_1F7
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGG
ATGACCTGTGGGGGAAACAACATTGAAAGTAAAACTGTGCATTGGTACCAGCAGAAGC
CGGGCCAGGCCCCTGTGCTGGTCGTCTACAATGATAACGTCCGGCCCTCAGGGATCC
CTGCGCGATTCTCTGGCTCCAACTCCGGCAACACGGCCACCCTGACCATCAACAGGGT
CGAAGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGACTCCAGTAGAGATCAA
GGGGTATTCGGCGGAGGGACCAAGCTGACCGTC (SEQ ID NO: 97)
Her2_S1 R2A_CS_1 D11
VH with CDR1 , CDR2 and CDR3 underlined
GGAGGCCTGGGTCCTCGGTGAGGGTCTCCTGCACGGCTTCTGGAGACACCTCCAGCA
GCTTTACCGTCAACTGGCTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGAG
GGATCACCCCTATGTTTGGCACTGCAAACTACGCACAGATGTTCGAGGACAGAGTCAC
GATAACCGCGGACGAAATGGAACTGAGTGGCCTGACATCTGAGGACACGGCCGTGTAT
TTTTGTGCGACAGGCCCCTCCGATTACGTTTGGGGGAGTTATCGTTTCCTTGACACCTG
GGGGCGGGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO: 98)
Her2_S1 R2A_CS_1 D11
VL with CDR1 , CDR2 and CDR3 underlined
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGCGGCCCCAGGACAGGAGGTCTCC
ATCTCCTGCTCTGGAGCCAGATCCAACGTTGGGGGTAATTATGTTTCCTGGTACCAACA
CCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGA
TGCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGG
AGTCCAGACTGAGGACGAGGCCGATTATTACTGCGCAACATGGGATAGCAGCCTGAGC
GCTGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 99)
Her2_S1R2C_CS_1D3
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAGGAGGCCTGGGTCCTCGGTGAGG
ATCTCCTGCACGGCTTCTGGAGACACCTCCAGCAGCTTTACCGTCAACTGGGTGCGAC
AGGCCCCTGGACAAGGTCTTGAGTGGATGGGAGGGATCACCCCTATGTTTGGCACTGC
AAACTACGCACAGGTGTTCGAGGACAGAGTCACAATAATCGCGGACGAGATGGAACTG
AGTGGCCTGACATCTGAGGACACGGCCGTGTATTTCTGTGCGACAGGCCCCTCCGATT
ACGTTTGGGGGAGTTATCGTTTCCTTGACAACTGGGGCAGGGGCACCCTGGTCACCGT
CTCGAGT (SEQ ID NO: 100) Her2_S1R2C_CS_1D3
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCTGCGGCCCCAGGGCAGAAGGTCACC
ATCTCCTGCTCTGGAGGCAGGTCCAGCATTGGGAATAATTATGTGTCCTGGTATCAACA
CCTCCCAGGAACAGCCCCCAAACTCCTCATCTATGACAATAATCAGCGACCCTCAGGG
ATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCG
GACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGA
GTGCTGTGGTGTTTGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 101 )
Her2_S1R2C_CS_1H12
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG
ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGAACTGGGTCCGC
CAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTTCTGGTAATACCA
TATTCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAGTGCCAAGAAT
TCAGTGTCTCTGCAGATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTG
CTTCCTACTACTCCTACTACTACGGTATGGACGCCTGGGGCCAGGGGACAATGGTCAC
CGTCTCGAGTTCGAGT (SEQ ID NO: 102)
Her2_S1R2C_CS_1H12
VL with CDR1 , CDR2 and CDR3 underlined
TCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCG
GAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCAT
CTATAGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCT
GGCACCTCAGCCTCCCTGGCCATCAGTGGGCTGCGGTCCGAGGATGAGGCTGATTATT
ACTGTGCAGCATGGGATTACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGG
TCACCGTCCTA (SEQ ID NO: 103)
Her2_S1R2A_CS_1D3
VH with CDR1 , CDR2 and CDR3 underlined
GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG
GTCTCCTGCAAGGCTTCTGGGTACAGCTTCACCGCCTTCTATATTCACTGGGTGCGACA
GGCCCCTGGACAAGGCCTTGAGTATTTGGGATGGATCGACCCTAATACTGGTGCCACA
AAATATGCACAGCGCTTTCAGGGCAGGGTCATCATGACCTGGGACACGTCCATCACCA
CAGCCACCATGGAACTGAGCAGGCTGACGTCTGACGACTCGGCCGTCTACTACTGTGT
GAGAGATTTGCGGGAGTGGGGCTACGAATTGTCCGTTGAGTATTGGGGCAGAGGAAC
CCTGGTCACCGTCTCGAGT (SEQ ID NO: 104)
Her2_S1R2A_CS_1D3
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACC
ATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCA
GCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGG
GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTG
GGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAG
TGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 105) Her2_S1 R3B2_BMV_1 E1
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGGAGACTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAG CCTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCAGTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGTTTATACGGTACGATGGAAGTAGT GAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT GGAAGAACGCTGGAGTCTAGTTTGTGGGGCAAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 106)
Her2_S1 R3B2_BMV_1 E1
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACC
ATTTCCTGCTCTGGAAGCACCTCCAACATTGGGAATAATTATGTCTCCTGGTACCAACA
GCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGG
GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAACTCAGCCTCCCTGGACATCAGTG
GGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAG
TGAATTTCTCTTCGGAACTAGGACCAAGCTGACCGTCCTA (SEQ ID NO: 107)
Her2_S1R3C1_CS_1D3
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGCAGGAGTCGGGTCCAGGACTGGTGAAGCCCTCGCAGACCTTGTCA
CTCACCTGTGGCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGA
TCAGGCAGTCCCCAACGAGAGGCCTTGAGTGGCTGGGAAGGACATATTACAGGTCCAG
TTGGTATCATAACTATGCACCTTCTATGAACAGTCGATTAACCATCATCGCAGACACATC
CAAAAACCAGTTCTCTTTGCAACTGAACTCTGTGACTCCCGAGGACACGGCTGTATATT
ACTGTGCAAGCGGGTGGGCCTTTGATGTCTGGGGCAGGGGAACCCTGGTCACCGTCT
CGAGT (SEQ ID NO: 108)
Her2_S1R3C1_CS_1D3
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCA
TCTCCTGCACTGGAACCAGCAGTGACGTTGGTGCTTATGACTTTGTCTCCTGGTACCAA
CAGCACCCTGGCAAAGCCCCCAAACTCATGATTTATGAGGTCAATAAGCGGCCCTCAG
GGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTC
TGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAAG
AATTTGCTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 109)
Her2_S1 R3B2_DP47_1 E8
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCC
AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCGAGACAGTCGGGCGCGGACTGGTACTTCGATCTCTGGGGCCGAGGCACCCTGGTC
ACCGTCTCGAGT (SEQ I D NO: 110) Her2_S1 R3B2_DP47_1 E8
VL with CDR1 , CDR2 and CDR3 underlined
CAGGCTGTGCTGACTCAGCCGTCCGCAGTTTCTGGGGCCCCAGGGCAGAGGGTCACC
ATCTCCTGCACTGGGACCAGCTCCAACATCGGGACAAACTATCTTGTACACTGGTATCA
GCAACGTCCAGGAACAGCCCCCCAACTCCTCGTCTCTGGTAACAACACTCGACCCTCT
GGGGTCACTGACCGGTTCTCTGTCTCCAAGTCTGCCACTTCAGCCTCCCTGGCCATCA
CTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGACCTATGACATCAACTT
GAGGGTTTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 111 )
Her2_S1 R3B2_BMV_1 G2
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAG
GTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACA
GGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACA
AACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA
CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGC
GAGAGTCCCGGGCGTAAGTGGGAGCTATCCAGACTACTACTACATGGACGTCTGGGG
CAAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 112)
Her2_S1 R3B2_BMV_1 G2
VL with CDR1 , CDR2 and CDR3 underlined
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCAC
CATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAG
CCAGGGAAAGCTCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCC
CATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCT
GCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTT
CGGCGGAGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 113)
Her2_S1 R3B2_BMV_1 H5
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCAGGCCTGGAGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCGGGATTCTCCTTCAGTGACTACTACATGACCTGGATCCGCCA
GATTCCAGGGAAGGGGCTGGAGTGGGTGGCAGTTATATGGAATGATGGAAGTGATAGA
TACTATGCAGACTCCGTGAAGGGCCGATTCACCATTTCCAGAGACAATTCCAAGAACAC
GCTGTTTCTGCAAATGAGCAGCCTGAGAGACGAGGACACGGCTCTATATTACTGTGTG
AGAGGGGGACCAACAGCTTCAAGCGGATTTGACTACTGGGGCCGAGGCACCCTGGTC
ACCGTCTCGAG (SEQ ID NO: 114)
Her2_S1 R3B2_BMV_1 H5
VL with CDR1 , CDR2 and CDR3 underlined
TCGTCTGAGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCA TCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCTA CAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAG GGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCT GGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGAGCA CTCGAGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 115) Her2_S1 R3C1_CS_1 A6
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTAAGGGTTTTGGATACAATTTTCGCAGCGCCTGGATCGGCTGGGTGCGCC
AGATGCCCGGCAAAGGCCTGGAGTGGATGGGGGTCATCTATCCTGGTGACTCTGATGT
CAGATACAGTCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGT
ACCGCCTACCTGCAGTGGAGCAGCCTGAAAGCCTCGGACACCGCCATGTATTATTGTA
CGAGACCCGTAGGGCAGTGGGTGGACTCTGACTATTGGGGCAAGGGAACCCTGGTCA
CCGTCTCGAGT (SEQ ID NO: 116)
Her2_S1R3C1_CS_1A6
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGTTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGACAGAGGGTCACC ATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAACTAATACTGTGAACTGGTACCAGCA GCTTCCAGGAACGGCCCCCAAACTCCTCATCTATACTAGTAATCAGCGGCCCTCAGGG GTCCCTGCCCGCTTCTCTGCCTCCAACTCTGGCACCTCAGCCTCCCTGGCCATCAGTG GGCTCCGGTCCGAGGATGAGGCTGATTATTATTGTGCAGCGTGGGATGACAAGTTGAG TGGTGCGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 117)
Her2_S1 R3B2_DP47_1 C9
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCC
AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCGAGATGGAGGCCTCTTCTAGACTACCACTTTGACCAATGGGGCCAAGGGACAATGG
TCACCGTCTCGAGT (SEQ ID NO: 118)
Her2_S1 R3B2_DP47_1 C9
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGACAGACGGTAACAA
TCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAGTGTTGTTAATTGGTACCAGCAG
TTCCCAGGAACGGCCCCCAAAGTCCTCGTCTATAGTAACACTCAGCGGCCCTCAGGGG
TCCCTGACCGATTCTCTGGCTCCAGGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGG
GCTCCAGTCTGAGGATGAGGCTGATTATTACTGTTTAGCATGGGATGCCAGCCTGAATG
GTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 119)
Her2_S1 R3B2_DP47_1 E10
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCC
AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCGAGAGGATACAGTGGCTACGATGACCCTGACTCCTGGGGGAGAGGGACCACGGTC
ACCGTCTCGAGT (SEQ ID NO: 120) Her2_S1R3B2_DP47_1E10
VL with CDR1, CDR2 and CDR3 underlined
CACGTTATACTGACTCAACCGCCCTCAACGTCTGGGACCCCCGGGCAGACGGTCACCA
TCTCTTGTTCTGGGAGCAGCTCCAACATCGGAAGTCATTATGTATACTGGTACCAGCAG
CTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGG
TCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGG
GCTCCGGTCCGAGGATGAGACTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGT
GGTCGAGTCTTCGGAACTGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 121 )
Her2_S1 R3C1_CS_1 B10
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAG GTCTCCTGCAAGGCTTCTGGAGGCACCATCAGCAACTATGCTATCAGTTGGGTGCGGC TGGCCCCTGGACAAGGTCTTGAGTGGATGGGAAGTATCGTCCCTCTTCATGGGACAAC AAACTTCGCACAGAAATTCCAGGGCAGAGTCACGATCACCGCGGACGAGTCCACGAGC ACATCCTACATGGAGGTGAACGTCCTGACATATGAAGACACGGCGATGTATTATTGTGC GTCTCTCAATTGGGGCTACTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 122)
Her2_S1 R3C1_CS_1 B10
VL with CDR1 , CDR2 and CDR3 underlined
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCA
TCTCCTGCACCGGCAGTAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCA
GCGCCCGGACAGTGCCCCCACCACTGTGATCTATGAGGATAATCGAAGATCCTCTGGA
GTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCAGCAT
CTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCCTATGATAGTAGC
GGTCATGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 123)
Her2_S1 R3A1_BMV_1 F3
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGGAGTCTGGGGAAGGCCTGGTCAAGCCTGGGGGGTCCCTGAG
ACTCTCCTGTACAGCCTCTGGATTCACCTTCAGGAGTTATAGCTTGAACTGGGTCCGCC
AGGCTCCAGGGCAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTACTAGTACTTACAT
ATACTACGCAGACTCGGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAAC
ACACTGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACAGCTGCATATTACTGTGT
TAGACTGGGATCTGGTGGGGGATATTTTCCTGACTACTGGGGCAGGGGCACCCTGGTC
ACCGTCTCGAGT (SEQ ID NO: 124)
Her2_S1 R3A1_BMV_1 F3
VL with CDR1 , CDR2 and CDR3 underlined
TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGA
TCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCC
AGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCA
GACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTC
AGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGT
GGIATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 125) Her2_S1R3B1_BMV_1G11
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTACCTATGCCATGAGTTGGGCCCGCC AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGGTGATGGTGGAAGAA TTCTCGATGCAGACTCCGCGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA CACGCTGTATCTGCAAATGAACGGCCTGAGAGTCGAGGACACGGCCCTTTATTACTGT GCGAGAGCGGACGGTAACTACTGGGGCAGGGGGACAATGGTCACCGTCTCTTCA (SEQ ID NO: 126)
Her2_S1 R3B1_BMV_1G11
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCA TCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAA CAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAG GGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCT GGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGAGCA CTCGAGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 127)
Her2_S1 R3A1_BMV_1G4
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG
GTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACA
GGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCTGGCAATGGTAACAC
AAAATATTCACAGAAGTTCCAGGGCAGAGTCACCATTACCAGGGACACATCCGCGAGC
ACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTG
CGAGAGGGAGGAGCTATGGCCACCCGTACTACTTTGACTACTGGGGCCAGGGAACCC
TGGTCACCGTCTCGAGT (SEQ ID NO: 128)
Her2_S1 R3A1_BMV_1G4
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCA TCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAA CAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAG GGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCT GGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGAGCA CTCGAGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 129)
Her2_S1 R3B1_BMV_1 H11
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAG
ACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGGATGCACTGGGTCCGC
CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGGTA I I I I I I ATGATGGAGGTAATA
AATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG
CGAGAGATAGGGGCTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCG
TCTCCTCA (SEQ ID NO: 130) Her2_S1 R3B1_BMV_1 H11
V1 with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGACAGAGGGTCACC
ATCTCCTGCACTGGGAGAAGCTCCAACATCGGGGCGGGTCATGATGTACACTGGTACC
AGCAACTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTGACAGCAATCGGCCCTC
AGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCTGGCACCTCAGCCTCCCTGGCCATC
ACTGGGCTCCAGGCTGAAGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCC
TGAGGGGTTCGGTATTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 131 )
Her2_S1R3A1_CS_1B9
VH with CDR1, CDR2 and CDR3 underlined
AAGGTGCAGCTGGTGCAGTCTGGGACAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTCAGGGTTCTGGATACAGGTTTAGTAGTGACTGGATTGCCTGGGTGCGCC
AGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATTGTCTATCCTGGTGACTCTGATAC
CAGATATAGCCCGTCCTTCCAAGGCCAAGTCACCATCTCAGCCGACAAGTCCATCAGTA
CTGCCTACCTGCAGTGGAGCGGCCTGAAGGCCTCGGACACCGCCAAGTATTACTGTGC
GAGAGTGCAACAGGCAGTGGGAGCTAAAGGTTATGCTATGGACGTCTGGGGCAAGGG
AACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 132)
Her2_S1 R3A1_CS_1 B9
VL with CDR1, CDR2 and CDR3 underlined
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACAC
TCACTTGTGGCTTGAGCTCTGGCTCAGTCTCTACCAGTTACTACCCCAGCTGGTACCGG
CAGACCCCAGGCCAGGCTCCACACACACTCATTCACAACACAAAGATTCGCTCCTCTG
GGGTCCCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAATGCTGCCCTCACCATCAC
GGGGGCCCAGGCAGATGATGAATCTGATTATTACTGTCTTTTGTATATGGGTAGCGGCA
TTTACGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 133)
Her2_S1 R3B1_BMV_1 H9
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGCAGGAGTCGGGCGCAGGACTGGTGAAGCCTTCGGGGACCCTGTCC
CTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTGGTAACTGGTGGAGTTGGGTCC
GCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTCTCATAGTGGGAGCA
CCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAAC
CAGTTCTCCCTGAACCTGAGTTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTG
CGAGAGTAAGGGGTACGGTGGGGGATACACGGGGACCTGACTACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 134) Her2_S1 R3B1_BMV_1 H9
VL with CDR1 , CDR2 and CDR3 underlined
TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGA
TCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCC
AGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCA
GACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTC
AGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGT
GGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 135)
Her2_S1 R3A1_CS_1 B10
VH with CDR1 , CDR2 and CDR3 underlined
GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGG
GTCTCCTGCAAGGGTTCTGGAAACACCTTCACCGGCCACTACATCCACTGGGTGCGAC
AGGCCCCTGGACAAGGACTTGAGTGGCTGGGATGGATCGACCCTAACACTGGTGACAT
ACAGTATTCAGAAAACTTTAAGGGCTCGGTCACCTTGACCAGGGACCCATCCATCAACT
CAGTCTTCATGGACCTGATCAGGCTGACATCTGACGACACGGCCATGTATTACTGTGC
GAGAGAAGGTGCCGGGCTCGCCAACTACTATTACTACGGTCTGGACGTCTGGGGCCG
AGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 136)
Her2_S1 R3A1_CS_1 B10
VL with CDR1 , CDR2 and CDR3 underlined
CAGACTGTGGTGCTCCAGGAGCCTTCGTTCTCAGTGTCCCCTGGGGGGACAGTCACAC
TCACTTGTGGCTTGAACTTTGGCTCAGTCTCTACTGCTTACTACCCCAGTTGGTACCAG
CAGACCCCAGGCCAAGCTCCACGCACGCTCATCTACGGCACAAATATTCGTTCCTCTG
GGGTCCCGGATCGCTTCTCTGGCTCCATCGTAGGGAACAAAGCTGCCCTCACCATCAC
GGGGGCCCAGACAGAAGATGAGTCTGATTATTATTGTGCGCTGTATATGGGTAGTGGC
ATGCTCTTCGGCGGCGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 137)
Her2_S1R3B1_BMV_1C12
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG
ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGC
CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATTA
AATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG
CGCGAACTGGTGAATATAGTGGCTACGATACGAGTGGTTACAGCAATTGGGGCCAAGG
CACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 138) Her2_S1R3B1_BMV_1C12
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACC
ATCTCTTGTTCTGGAAGCAGCTCCAACATCGGGAGTAACACTGTAAACTGGTACCAGCG
ACTCCCAGGAGCGGCCCCCCAACTCCTCATCTACAATAATGACCAGCGGCCCTCAGGG
ATCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGCTCCCTGGTCATCAGTG
GGCTCCAGTCTGAAGATGAGGCTGATTACTACTGTGCGTCATGGGATGACAGTCTGAA
TGGTCGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 139)
Her2_S1R3C1_BMV_1H11
VH with CDR1 , CDR2 and CDR3 underlined
GGGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAG
ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAACATGAACTGGGTCCGCC
AGGCTCCAGGGAAGGGACTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGACGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT
GCGAAAGATACCAGTGGCTGGTACGGGGACGGTATGGACGTCTGGGGCCGGGGAACC
CTGGTCACCGTCTCGAGT (SEQ ID NO: 140)
Her2_S1 R3C1_BMV_1 H11
VL with CDR1 , CDR2 and CDR3 underlined
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCAC
CATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAG
CCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCC
CATCAAGGTTCAGCGGCAGTGGATCAGGGACAGATTTCACTCTCACCATCAGCAGCCT
GCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTT
CGGCGGAGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 141 )
Her2_S1R3B1_BMV_1A10
VN with CDR1 , CDR2 and CDR3 underlined
CAGATGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCC
AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATTAA
ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA
CACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACACGGGCGTTTATTACTGTTCG
AAAGATCGCTATAGCAGTGGCTGGTACAGCTCCGATGCTTTTGATATTTGGGGCCGAG
GGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 142)
Her2_S1R3B1_BMV_1A10
VL with CDR1 , CDR2 and CDR3 underlined
TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCA
CATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGG
ACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGAC
CGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGG
CGGAAGATGAGGCTGACTATTACTGTCATTCCCGGGACAGCAGTGGTAACCATGTGCT
TTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 143) Her2_S1 R3A1_CS_1 D11
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGAGAGTCTCTGAAG
ATCTCCTGTAAGGGCTCTGGATACACCTTTACCAACCACTGGATCGCCTGGGTGCGCC
AGATGCCCGGGAAAGGCCTGGAGTGGATGGGCATCATCTATCCTGGTGACTCTGAAAC
GAGGTACAGCCCGTCCTTCCAAGGCCACGTCACCATCTCAGCCGACAAGTCCATCAGT
ACCGCCTATTTGCAGTGGAGCACCCTGAAGGACTCGGACTCCGCCATGTACTTCTGTG
TGAGACAGGCCCGTGGCTGGGACGACGGACGGGCTGGATATTATTATTCCGGTATGGA
CGCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 144)
Her2_S1R3A1_CS_1D11
VL with CDR1 , CDR2 and CDR3 underlined
CAGGCTGTGGTGCTCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACAC
TCACCTGTGGCTTGCGCTCTGGGTCAGTCTCTACTAGTCACTACCCCAGCTGGTACCA
GCAGACCCCAGGCCAGGCTCCACGCACGCTCATTTACAGCACAAACACTCGCTCTTCT
GGGGTCCCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCA
CGGGGGCCCAGGCAGATGATGAATCTAATTATTACTGTATGCTATACATGGGCAGTGG
CATGTATGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 145)
Her2_S1 R3C1_DP47_1 H1
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCC
AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCGAGAGTCAGCGGGAGCCACTTTCCATTCTTTGACTCCTGGGGCCAGGGGACAATG
GTCACCGTCTCGAGT (SEQ ID NO: 146)
Her2_S1 R3C1_DP47_1 H1
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGA
ATTACCTGTGGGGGAGACAAGATTGGACATAAAAGTGTGCATTGGTATCAGCAGAAGC
CAGGCCAGGCCCCTGTGTTGCTCGTCTATGATGATAGGAAGCGGCCCTCAGGGATCCC
TGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTC
GAGGCCGGGGATGAGGCTGCCTATCACTGTCAGGTGTGGGATAGAAGTAGTGACCCTT
ATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 147) Her2_S1 R3A1_CS_1 B12
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGGTGCAATCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAG
GTCTCTTGTCAGGCTTCTGGATACACCTTCAGCGGGCACTATATGCACTTGGTGCGACA
GGCCCCTGGACAAGGGCTTGAGTGGATGGGGTGGATCCACCCTACCAGTGGTGGCAC
AACCTATGCACAGAAGTTTCAGGGCCGGGTCGTTATGACCAGGGACACGTCCATCAGC
ACAGCCTACATGGAACTGAGTAGGCTGACATCTGACGACACGGCCGTGTATTACTGTG
CAAGAATGTCCCAAAACTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACC
GTCTCGAGT (SEQ ID NO: 148)
Her2_S1R3A1_CS_1B12
VL with CDR1 , CDR2 and CDR3 underlined
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACC
ATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTAAACTGGTACC
AACAATTTCCAGGAACAGCCCCCAAAATTATCGTCTATGGCGATCGGCCCTCAGGGGC
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCAATCACTGGA
CTCCGGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTGGGACAGTCGCCTGAGTA
GTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 149)
Her2_S1 R3B1_BMV_1 H5
VH with CDR1 , CDR2 and CDR3 underlined
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAG
ACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTGGCTATGGCATGCACTGGGTCCGC
CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATCTGTACGGAACGATGGAAGTAAT
ACATACTACACAGACTCCGTGAAGGACCGATTCACCATCTCCAGAGACAACACCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT
GCCAAGTCGAGAAGAGTGATGTATGGCACCTCCTATTACTTTGACTACTGGGGCAGAG
GCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 150)
Her2_S1 R3B1_BMV_1 H5
VL with CDR1 , CDR2 and CDR3 underlined
TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGA
TCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCC
AGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCA
GACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTC
AGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGT
GGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 151 ) Her2_S1 R3A1_DP47_1 A6
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCC
AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCGAGAGATCTGGGAATAGACCCCCTTTGGAGTGGTTATTACACACCCCTTGACTATTG
GGGCCGAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 152)
Her2_S1 R3A1_DP47_1 A6
VL with CDR1 , CDR2 and CDR3 underlined
CACGTTATACTGACTCAACCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACC
ATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTCCGTTAGCTGGTACCAGCA
GCTCCCAGGAACGGCCCCCAAACTCCTCATGTATACTAACAATCAGCGGCCCTCAGGG
GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTG
GGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCGACATGGGATGCCAGCCTGAA
TACTTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 153)
Her2_S1 R3B1_DP47_1 E1
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCC
AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCA
CATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAA
CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGT
GCGAGAGGCGGGAGTGGGAGTGACTACTGGGGCCAGGGGACAATGGTCACCGTCTC
GAGT (SEQ ID NO: 154)
Her2_S1 R3B1_DP47_1 E1
VL with CDR1 , CDR2 and CDR3 underlined
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGGGTCTCCGGGGAAGACGGTAACCA
TCTCCTGCACCCGCAGCAGTGGCTACATTGACAGCAAGTATGTGCAGTGGTACCAGCA
GCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGAGGATAACCGAAGACCCTCTGG
GGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACC
ATCTCTGGACTGGAGACTGAGGACGAGGCTGACTATTACTGTCAGTCTTATGATGACAC
CAATGTGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 155) Her2_S1R3B1_BMV_1A1
VH with CDR1 , CDR2 and CDR3 underlined
GAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGGAGCCTGGGGCCTCAGTGAAG
GTCTCCTGCAAGGCCTCTGGTTACGACTTTTCCAACTATGGTTTCAGCTGGGTGCGCCA
GGCCCCTGGACAAGGTCTTGAGTGGATGGGATGGATCAGCTCTTATAATGGTTACACA
AACTATGCACAGAGACTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA
CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACAGCTGTCTATTACTGTGC
GAGAGATCGAGGACTTGGAAACTGGTACTTCGATCTCTGGGGCCAAGGCACCCTGGTC
ACCGTCTCGAGT (SEQ ID NO: 156)
Her2_S1R3B1_BMV_1A1
VL with CDR1 , CDR2 and CDR3 underlined
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCA TCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAA CAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAG GGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCT GGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGAGCA CTCGAGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 157)
>HER018_CDS atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagatattcagatgacccagagcc cgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcgcgagccaggatgtgaacaccgcggtggcgt ggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatagcgcgagctttctgtatagcggcgtgccgagccgcttta gcggcagccgcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgccagcagca ttataccaccccgccgacctttggccagggcaccaaagtggaaattaaacgcaccgggggtggaggctctggtggcggtggct ctggcggaggtggatccggtggcggcggatctgaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggca gcctgcgcctgagctgcgcggcgagcggctttaacattaaagatacctatattcattgggtgcgccaggcgccgggcaaaggcc tggaatgggtggcgcgcatttatccgaccaacggctatacccgctatgcggatagcgtgaaaggccgctttaccattagcgcgga taccagcaaaaacaccgcgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcagccgctggggcg gcgatggcttttatgcgatggattattggggccagggcaccctggtgaccgtgagcagtgatcaggagcccaaatcttgtgacaa aactcacacatctccaccgtgctcagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacacc ctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtac gtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtc ctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcg agaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgacc aagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagc cggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctc cctgtctccgggtaaatga (SEQ ID NO: 158)
>HER018_Protein_leader-stop
MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT
KVEIKRTGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT
YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV
YYCSRWGGDGFYAMDYWGQGTLVTVSSDQEPKSCDKTHTSPPCSAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 159) >HER018_2h7_Leader_CDS atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagagga (SEQ ID NO: 160)
>HER018_2h7_Leader_Protein MDFQVQIFSFLLISASVIMSRG (SEQ ID NO: 161 )
>HER018_VL_CDS
Gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcgcgagccag gatgtgaacaccgcggtggcgtggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatagcgcgagctttctgtat agcggcgtgccgagccgctttagcggcagccgcagcggcaccgattttaccctgaccattagcagcctgcagccggaagatttt gcgacctattattgccagcagcattataccaccccgccgacctttggccagggcaccaaagtggaaattaaacgcacc (SEQ ID NO: 162)
>HER018_VL_Protein
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT (SEQ ID NO: 163)
>HER018_G4Sx4_Linker_CDS gggggtggaggctctggtggcggtggctctggcggaggtggatccggtggcggcggatct(SEQ ID NO: 164)
>HER018_G4Sx4_Linker_Protein GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 165)
>HER018_VH_CDS gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggcttt aacattaaagatacctatattcattgggtgcgccaggcgccgggcaaaggcctggaatgggtggcgcgcatttatccgaccaac ggctatacccgctatgcggatagcgtgaaaggccgctttaccattagcgcggataccagcaaaaacaccgcgtatctgcagatg aacagcctgcgcgcggaagataccgcggtgtattattgcagccgctggggcggcgatggcttttatgcgatggattattggggcc agggcaccctggtgaccgtgagcagt(SEQ ID NO: 166)
>HER018_VH_Protein
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS S (SEQ ID NO: 167)
>HER018_CSCS_Hinge_CDS gagcccaaatcttgtgacaaaactcacacatctccaccgtgctca (SEQ ID NO: 168)
>HER018_CSCS_Hinge_Protein EPKSCDKTHTSPPCS (SEQ ID NO: 169)
>HER018_Fc_Stop_CDS gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga(SEQ ID
NO: 170) >HER018_Fc_Stop_Protein
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 171 )
>HER026_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctgggtct gaggtgaggaggcctgggtcctcggtgagggtctcctgcacggcttctggagacacctccagcagctttaccgtcaactggctgc gacaggcccctggacaaggtcttgagtggatgggagggatcacccctatgtttggcactgcaaactacgcacagatgttcgagg acagagtcacgataaccgcggacgaaatggaactgagtggcctgacatctgaggacacggccgtgtatttttgtgcgacaggc ccctccgattacgtttgggggagttatcgtttccttgacacctgggggcgggggaccacggtcaccgtctcgagtggaggcggcg gttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtgctgactcagccgtcctcagtgtctgcggccccaggac aggaggtctccatctcctgctctggagccagatccaacgttgggggtaattatgtttcctggtaccaacacctcccaggaacagcc cccaaactcctcatttatgacaataataagcgaccctcagggatgcctgaccgattctctggctccaagtctggcacgtcagccac cctgggcatcaccggagtccagactgaggacgaggccgattattactgcgcaacatgggatagcagcctgagcgctgtggtctt cggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac tggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 172)
>HER026_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGSEVRRPGSSVRVSCTASGDTSSSFTVNWLRQA
PGQGLEWMGGITPMFGTANYAQMFEDRVTITADEMELSGLTSEDTAVYFCATGPSDYVW
GSYRFLDTWGRGTTVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSAAPGQEVSISCS
GARSNVGGNYVSWYQHLPGTAPKLLIYDNNKRPSGMPDRFSGSKSGTSATLGITGVQTED
EADYYCATWDSSLSA WFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 173)
>HER027_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtgcagtctgggtct gaggtgaggaggcctgggtcctcggtgaggatctcctgcacggcttctggagacacctccagcagctttaccgtcaactgggtgc gacaggcccctggacaaggtcttgagtggatgggagggatcacccctatgtttggcactgcaaactacgcacaggtgttcgagg acagagtcacaataatcgcggacgagatggaactgagtggcctgacatctgaggacacggccgtgtatttctgtgcgacaggc ccctccgattacgtttgggggagttatcgtttccttgacaactggggcaggggcaccctggtcaccgtctcgagtggaggcggcgg ttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgactcagccaccctcagtgtctgcggccccagggca gaaggtcaccatctcctgctctggaggcaggtccagcattgggaataattatgtgtcctggtatcaacacctcccaggaacagcc cccaaactcctcatctatgacaataatcagcgaccctcagggattcctgaccgattctctggctccaagtctggcacgtcagccac cctgggcatcaccggactccagactggggacgaggccgattattactgcggaacatgggatagcagcctgagtgctgtggtgttt ggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcc cagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctga ggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcata atgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggact ggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 174)
>HER027_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGSEVRRPGSSVRISCTASGDTSSSFTVNWVRQA
PGQGLEWMGGITPMFGTANYAQVFEDRVTIIADEMELSGLTSEDTAVYFCATGPSDYVWG
SYRFLDNWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSAAPGQKVTISCSG
GRSSIGNNYVSWYQHLPGTAPKLLIYDNNQRPSGIPDRFSGSKSGTSATLGITGLQTGDEA
DYYCGTWDSSLSAWFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK (SEQ ID NO: 175)
>HER028_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaagtgcagctggtgcagtctggggc tgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctgggtacagcttcaccgccttctatattcactgggtgcg acaggcccctggacaaggccttgagtatttgggatggatcgaccctaatactggtgccacaaaatatgcacagcgctttcagggc agggtcatcatgacctgggacacgtccatcaccacagccaccatggaactgagcaggctgacgtctgacgactcggccgtcta ctactgtgtgagagatttgcgggagtggggctacgaattgtccgttgagtattggggcagaggaaccctggtcaccgtctcgagtg gaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgactcagccaccctcagcgtctggg acccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaattatgtatactggtaccagcagctcc caggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctgg cacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgacagcctgagt ggttgggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgc ccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg gaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga (SEQ ID NO: 176)
>HER028_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGASVKVSCKASGYSFTAFYIHWVRQAP
GQGLEYLGWIDPNTGATKYAQRFQGRVIMTWDTSITTATMELSRLTSDDSAVYYCVRDLRE
WGYELSVEYWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTPGQRVTISC
SGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSED
EADYYCAAWDDSLSGWVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 177)
>HER029_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggc tgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgc gacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcag ggctgggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggcc gtgtattactgtgcgagagattctactatggccccaggtgcttttgatatctggggccgaggcaccctggtcaccgtctcgagtgga ggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgactcagccaccctcggtgtcagtggcc ccaggacagacggccaggatgacctgtgggggaaacaacattgaaagtaaaactgtgcattggtaccagcagaagccgggc caggcccctgtgctggtcgtctacaatgataacgtccggccctcagggatccctgcgcgattctctggctccaactccggcaacac ggccaccctgaccatcaacagggtcgaagccggggatgaggccgactattattgtcaggtgtgggactccagtagagatcaag gggtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccca ccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgga cccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcca aagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagc ctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactaca agaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatg a (SEQ ID NO: 178)
>HER029_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA
PGQGLEWMGWINPNSGGTNYAQKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCARD
STMAPGAFDIWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSVAPGQTARMT
CGGNNIESKTVHWYQQKPGQAPVLWYNDNVRPSGIPARFSGSNSGNTATLTINRVEAGD
EADYYCQVWDSSRDQGVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 179)
>HER030_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggg aggcttggtcaggcctggagggtccctgagactctcctgtgcagcctcgggattctccttcagtgactactacatgacctggatccg ccagattccagggaaggggctggagtgggtggcagttatatggaatgatggaagtgatagatactatgcagactccgtgaagg gccgattcaccatttccagagacaattccaagaacacgctgtttctgcaaatgagcagcctgagagacgaggacacggctctat attactgtgtgagagggggaccaacagcttcaagcggatttgactactggggccgaggcaccctggtcaccgtctcgagtggtg gaggcggttcaggcggaggtggcagcggcggtggcggatcgtctgagctgactcagcctgcctccgtgtctgggtctcctggac agtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtacctacaacacccaggcaaa gcccccaaactcatgatttatgagggcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggc ctccctgacaatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaaccaggagcactcgagttttcg gcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc agcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataat gccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacg cctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 180)
>HER030_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGGGLVRPGGSLRLSCAASGFSFSDYYMTWIRQIP
GKGLEWVAVIWNDGSDRYYADSVKGRFTISRDNSKNTLFLQMSSLRDEDTALYYCVRGGP
TASSGFDYWGRGTLVTVSSGGGGSGGGGSGGGGSSELTQPASVSGSPGQSITISCTGTS
SDVGGYNYVSWYLQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEA
DYYCSSYTTRSTRVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK (SEQ ID NO: 181 ) >HER031_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtcgggtc caggactggtgaagccctcgcagaccttgtcactcacctgtggcatctccggggacagtgtctctagcaacagtgctgcttggaa ctggatcaggcagtccccaacgagaggccttgagtggctgggaaggacatattacaggtccagttggtatcataactatgcacct tctatgaacagtcgattaaccatcatcgcagacacatccaaaaaccagttctctttgcaactgaactctgtgactcccgaggacac ggctgtatattactgtgcaagcgggtgggcctttgatgtctggggcaggggaaccctggtcaccgtctcgagtggaggcggcggtt caggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgactcagccaccctccgcgtccgggtctcctggacagt cagtcaccatctcctgcactggaaccagcagtgacgttggtgcttatgactttgtctcctggtaccaacagcaccctggcaaagcc cccaaactcatgatttatgaggtcaataagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctc cctgaccgtctctgggctccaggctgaggatgaggctgattattactgcagctcatatgcaggcagcaagaatttgcttttcggcgg agggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca cctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtca catgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcc aagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctg aatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagg gcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctg gtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct catgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 182)
>HER031_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLQESGPGLVKPSQTLSLTCGISGDSVSSNSAAWNWIRQ
SPTRGLEWLGRTYYRSSWYHNYAPSMNSRLTIIADTSKNQFSLQLNSVTPEDTAVYYCASG
WAFDVWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGSPGQSVTISCTGTS
SDVGAYDFVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEA
DYYCSSYAGSKNLLFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK (SEQ ID NO: 183)
>HER032_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctggggg aggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccg ccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaaggg ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagaggatacagtggctacgatgaccctgactcctgggggagagggaccacggtcaccgtctcgagtggaggc ggcggttcaggcggaggtggctctggcggtggcggaagtgcacacgttatactgactcaaccgccctcaacgtctgggacccc cgggcagacggtcaccatctcttgttctgggagcagctccaacatcggaagtcattatgtatactggtaccagcagctcccagga acggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctc agcctccctggccatcagtgggctccggtccgaggatgagactgattattactgtgcagcatgggatgacagcctgagtggtcga gtcttcggaactgggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccg tgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtg cataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccag gactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaa gccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcct gacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagg ggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 184) >HER032_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGY
SGYDDPDSWGRGTTVTVSSGGGGSGGGGSGGGGSAHVILTQPPSTSGTPGQTVTISCSG
SSSNIGSHYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDET
DYYCAAWDDSLSGRVFGTGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK (SEQ ID NO: 185)
>HER033_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtacagctgcagcagtcagggg ctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccatcagcaactatgctatcagttgggt gcggctggcccctggacaaggtcttgagtggatgggaagtatcgtccctcttcatgggacaacaaacttcgcacagaaattccag ggcagagtcacgatcaccgcggacgagtccacgagcacatcctacatggaggtgaacgtcctgacatatgaagacacggcg atgtattattgtgcgtctctcaattggggctactggggccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcgg aggtggctctggcggtggcggaagtgcacttaattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaa ccatctcctgcaccggcagtagtggcagcattgccagcaactatgtgcagtggtaccagcagcgcccggacagtgcccccacc actgtgatctatgaggataatcgaagatcctctggagtccctgatcggttctctggctccatcgacagctcctccaactctgcctccct cagcatctctggactgaagactgaggacgaggctgactactactgtcagtcctatgatagtagcggtcatgtggtcttcggcggag ggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat gcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaag acaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatg gcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcag ccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtca aaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccg tgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 186)
>HER033_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLQQSGAEVKKPGSSVKVSCKASGGTISNYAISWVRLAP
GQGLEWMGSIVPLHGTTNFAQKFQGRVTITADESTSTSYMEVNVLTYEDTAMYYCASLNW
GYWGRGTLVTVSSGGGGSGGGGSGGGGSALNFMLTQPHSVSESPGKTVTISCTGSSGSI
ASNYVQWYQQRPDSAPTTVIYEDNRRSSGVPDRFSGSIDSSSNSASLSISGLKTEDEADYY
CQSYDSSGHWFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK SEQ ID NO: 187)
>HER034_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggc agaggtgaaaaagcccggggagtctctgaagatctcctgtaagggttttggatacaattttcgcagcgcctggatcggctgggtgc gccagatgcccggcaaaggcctggagtggatgggggtcatctatcctggtgactctgatgtcagatacagtccgtccttccaagg ccaggtcaccatctcagccgacaagtccatcagtaccgcctacctgcagtggagcagcctgaaagcctcggacaccgccatgt attattgtacgagacccgtagggcagtgggtggactctgactattggggcaagggaaccctggtcaccgtctcgagtggaggcg gcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgttgacgcagccgccctcagcgtctgggaccccc ggacagagggtcaccatctcttgttctggaagcagctccaacatcggaactaatactgtgaactggtaccagcagcttccaggaa cggcccccaaactcctcatctatactagtaatcagcggccctcaggggtccctgcccgcttctctgcctccaactctggcacctcag cctccctggccatcagtgggctccggtccgaggatgaggctgattattattgtgcagcgtgggatgacaagttgagtggtgcggtgt tcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac tggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 188)
>HER034_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCKGFGYNFRSAWIGWVRQM
PGKGLEWMGVIYPGDSDVRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRPV
GQWVDSDYWGKGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTPGQRVTISCS
GSSSNIGTNTVNWYQQLPGTAPKLLIYTSNQRPSGVPARFSASNSGTSASLAISGLRSEDE
ADYYCAAWDDKLSGAVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 189)
>HER035_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctggggg aggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccg ccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaaggg ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagacagtcgggcgcggactggtacttcgatctctggggccgaggcaccctggtcaccgtctcgagtggaggcg gcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtgctgactcagccgtccgcagtttctggggccccag ggcagagggtcaccatctcctgcactgggaccagctccaacatcgggacaaactatcttgtacactggtatcagcaacgtccag gaacagccccccaactcctcgtctctggtaacaacactcgaccctctggggtcactgaccggttctctgtctccaagtctgccactt cagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagacctatgacatcaacttgagggtttgg gtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccacc gtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacc cctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggt gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacca ggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaa agccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcc tgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagg ggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 190)
>HER035_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQS
GADWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAQAVLTQPSAVSGAPGQRVTISCT
GTSSNIGTNYLVHWYQQRPGTAPQLLVSGNNTRPSGVTDRFSVSKSATSASLAITGLQAED
EADYYCQTYDINLRVWVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 191 )
>HER036_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagactgggg gaggcgtggtccagcctggggggtccctgagcctctcctgtgcagcgtctggattcaccttcagtagctatggcatgcagtgggtc cgccaggctccaggcaaggggctggagtgggtggcgtttatacggtacgatggaagtagtgaatactatgcagactccgtgaa gggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctg tgtattactgtggaagaacgctggagtctagtttgtggggcaagggaaccctggtcaccgtctcgagtggtggaggcggttcagg cggaggtggcagcggcggtggcggatcgcagtctgtgttgacgcagccgccctcagtgtctgcggccccaggacagaaggtc accatttcctgctctggaagcacctccaacattgggaataattatgtctcctggtaccaacagcacccaggcaaagcccccaaac tcatgatttatgatgtcagtaagcggccctcaggggtccctgaccgattctctggctccaagtctggcaactcagcctccctggacat cagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgagtgaatttctcttcggaactaggac caagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaac tcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaa ggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagcccc gagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaagg cttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg gactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccg tgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 192)
>HER036_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVETGGGWQPGGSLSLSCAASGFTFSSYGMQWVRQA
PGKGLEWVAFIRYDGSSEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGRTLE
SSLWGKGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSTSNIG
NNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCA
AWDDSLSEFLFGTRTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK (SEQ ID NO: 193)
>HER037_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtgcagtctggagc tgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggttacacctttaccagctatggtatcagctgggtgcg acaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggtaacacaaactatgcacagaagctccag ggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggc cgtgtattactgtgcgagagtcccgggcgtaagtgggagctatccagactactactacatggacgtctggggcaagggaaccct ggtcaccgtctcctcaggtggaggcggttcaggcggtggcagcggcggtggcggatcggacatccagatgacccagtctccttc caccctgtctgcatctattggagacagagtcaccatcacctgccgggccagtgagggtatttatcactggttggcctggtatcagca gaagccagggaaagctcctaaactcctgatctataaggcctctagtttagccagtggggccccatcaaggttcagcggcagtgg atctgggacagatttcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacaatatagtaattatccgc tcactttcggcggagggaccaagctggagatcaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccca ccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgga cccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcca aagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagc ctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactaca agaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatg a (SEQ ID NO: 194)
>HER037_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQA
PGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARV
PGVSGSYPDYYYMDVWGKGTLVTVSSGGGGSGGGSGGGGSDIQMTQSPSTLSASIGDRV
TITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPD
DFATYYCQQYSNYPLTFGGGTKLEIKRDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 195)
>HER038_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctggggg aggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccg ccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaaggg ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagatggaggcctcttctagactaccactttgaccaatggggccaagggacaatggtcaccgtctcgagtggagg cggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgactcagccaccctcagcgtctgggaccc ccggacagacggtaacaatctcttgttctggaagcagctccaacatcggaagtagtgttgttaattggtaccagcagttcccagga acggcccccaaagtcctcgtctatagtaacactcagcggccctcaggggtccctgaccgattctctggctccaggtctggcacctc agcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtttagcatgggatgccagcctgaatggttgggt gttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgt gcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccc tgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagg actggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagc caaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 196)
>HER038_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWR
PLLDYHFDQWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTPGQTVTISC
SGSSSNIGSSWNWYQQFPGTAPKVLVYSNTQRPSGVPDRFSGSRSGTSASLAISGLQSE
DEADYYCLAWDASLNGWVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 197)
>HER039_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagactgggg gaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatggcatgaactgggtcc gccaggctccagggaaggggctggagtgggtttcatacattagtagttctggtaataccatattctacgcagactctgtgaagggc cgattcaccatctccagagacagtgccaagaattcagtgtctctgcagatgaacagcctgagagacgaggacacggctgtgtat tactgtgcttcctactactcctactactacggtatggacgcctggggccaggggacaatggtcaccgtctcgagtggaggcggcg gttcaggcggaggtggctctggcggtggcggaagtgcactttcctatgtgctgactcagccaccctcagcgtctgggacccccgg gcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcagctcccaggaac ggcccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcag cctccctggccatcagtgggctgcggtccgaggatgaggctgattattactgtgcagcatgggattacagcctgagtggttgggtgt tcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac tggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 198)
>HER039_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVETGGGLVQPGGSLRLSCAASGFTFSSYGMNWVRQA
PGKGLEWVSYISSSGNTI FYADSVKGRFTISRDSAKNSVSLQMNSLRDEDTAVYYCASYYS
YYYGMDAWGQGTMVTVSSGGGGSGGGGSGGGGSALSYVLTQPPSASGTPGQRVTISCS
GSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDE
ADYYCAAWDYSLSGWVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 199)
>HER071_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagactgggg aaggcctggtcaagcctggggggtccctgagactctcctgtacagcctctggattcaccttcaggagttatagcttgaactgggtcc gccaggctccagggcaggggctggagtgggtctcatccattagtagtactagtacttacatatactacgcagactcggtgaaggg ccgattcaccatctccagagacgacgccaagaacacactgtatctgcaaatgaacagcctgagagccgaagacacagctgc atattactgtgttagactgggatctggtgggggatattttcctgactactggggcaggggcaccctggtcaccgtctcgagtggtgga ggcggttcaggcggaggtggcagcggcggtggcggatcgtctgagctgactcaggaccctgctgtgtctgtggccttgggacag acagtcaggatcacatgccaaggagacagcctcagaagctattatgcaagctggtaccagcagaagccaggacaggcccct gtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttcctt gaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaaccatgtggtattcgg cggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccca gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 200)
>HER071_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVETGEGLVKPGGSLRLSCTASGFTFRSYSLNWVRQAP
GQGLEWVSSISSTSTYIYYADSVKGRFTISRDDAKNTLYLQMNSLRAEDTAAYYCVRLGSG
GGYFPDYWGRGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQGDS
LRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC
NSRDSSGNHWFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK (SEQ ID NO: 201 )
>HER072_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtggagtctggggc tgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccagttatgatatcaactgggtgc gacaggcccccggacaaaggcttgagtggatgggatggatcaacgctggcaatggtaacacaaaatattcacagaagttcca gggcagagtcaccattaccagggacacatccgcgagcacagcctacatggagctgaggagcctgagatctgacgacacggc cgtgtattactgtgcgagagggaggagctatggccacccgtactactttgactactggggccagggaaccctggtcaccgtctcg agtggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgcagtctgtgctgactcagcctgcctccgtgtctggg tctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacaacac ccaggcaaagcccccaaactcatgatttatgagggcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctgg caacacggcctccctgacaatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaaccaggagcac tcgagttttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccc accgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgg acccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcac caggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcca aagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagc ctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactaca agaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatg a (SEQ ID NO: 202)
>HER072_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVESGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAP
GQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELRSLRSDDTAVYYCARGR
SYGHPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSITISCT
GTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAE
DEADYYCSSYTTRSTRVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 203)
>HER073_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtaaggtgcagctggtgcagtctgggac agaggtgaaaaagcccggggagtctctgaagatctcctgtcagggttctggatacaggtttagtagtgactggattgcctgggtgc gccagatgcccgggaaaggcctggagtggatggggattgtctatcctggtgactctgataccagatatagcccgtccttccaagg ccaagtcaccatctcagccgacaagtccatcagtactgcctacctgcagtggagcggcctgaaggcctcggacaccgccaagt attactgtgcgagagtgcaacaggcagtgggagctaaaggttatgctatggacgtctggggcaagggaaccctggtcaccgtct cgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagactgtggtgatccaggagccatcgttc tcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctaccagttactaccccagctggtaccgg cagaccccaggccaggctccacacacactcattcacaacacaaagattcgctcctctggggtccctgatcgcttctctggctccat ccttgggaacaatgctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtcttttgtatatgggtagcgg catttacgtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatg cccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctg caccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatct ccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc agcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaact acaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcag caggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga (SEQ ID NO: 204)
>HER073_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGKVQLVQSGTEVKKPGESLKISCQGSGYRFSSDWIAWVRQM
PGKGLEWMGIVYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSGLKASDTAKYYCARVQ
QAVGAKGYAMDVWGKGTLVTVSSGGGGSGGGGSGGGGSAQTWIQEPSFSVSPGGTVT
LTCGLSSGSVSTSYYPSWYRQTPGQAPHTLIHNTKIRSSGVPDRFSGSILGNNAALTITGAQ
ADDESDYYCLLYMGSGIYVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 205)
>HER074_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaagtgcagctggtgcagtctggggc tgaggtgaagaagcctggggcctcagtgagggtctcctgcaagggttctggaaacaccttcaccggccactacatccactgggt gcgacaggcccctggacaaggacttgagtggctgggatggatcgaccctaacactggtgacatacagtattcagaaaactttaa gggctcggtcaccttgaccagggacccatccatcaactcagtcttcatggacctgatcaggctgacatctgacgacacggccatg tattactgtgcgagagaaggtgccgggctcgccaactactattactacggtctggacgtctggggccgagggacaatggtcacc gtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagactgtggtgctccaggagccttc gttctcagtgtcccctggggggacagtcacactcacttgtggcttgaactttggctcagtctctactgcttactaccccagttggtacca gcagaccccaggccaagctccacgcacgctcatctacggcacaaatattcgttcctctggggtcccggatcgcttctctggctcca tcgtagggaacaaagctgccctcaccatcacgggggcccagacagaagatgagtctgattattattgtgcgctgtatatgggtagt ggcatgctcttcggcggcgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgc ccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg gaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga (SEQ ID NO: 206)
>HER074_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGASVRVSCKGSGNTFTGHYIHWVRQA
PGQGLEWLGWIDPNTGDIQYSENFKGSVTLTRDPSINSVFMDLIRLTSDDTAMYYCAREGA
GLANYYYYGLDVWGRGTMVTVSSGGGGSGGGGSGGGGSAQTWLQEPSFSVSPGGTVT
LTCGLNFGSVSTAYYPSWYQQTPGQAPRTLIYGTNIRSSGVPDRFSGSIVGNKAALTITGAQ
TEDESDYYCALYMGSGMLFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 207)
>HER075_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaagtgcagctggtgcagtctggggc tgaagtgaagaagcctggggcctcagtgaaggtctcttgtcaggcttctggatacaccttcagcgggcactatatgcacttggtgc gacaggcccctggacaagggcttgagtggatggggtggatccaccctaccagtggtggcacaacctatgcacagaagtttcag ggccgggtcgttatgaccagggacacgtccatcagcacagcctacatggaactgagtaggctgacatctgacgacacggccgt gtattactgtgcaagaatgtcccaaaactatgatgcttttgatatctggggccaagggacaatggtcaccgtctcgagtggaggcg gcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtgctgactcagccgtcctcagtgtctggggccccag ggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatgtaaactggtaccaacaatttccag gaacagcccccaaaattatcgtctatggcgatcggccctcaggggcccctgaccgattctctggctccaagtctggcacctcagc ctccctggcaatcactggactccgggctgaggatgaggctgattattactgccagtcctgggacagtcgcctgagtagttatgtcttc ggaactgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc agcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataat gccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacg cctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 208) >HER075_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGASVKVSCQASGYTFSGHYMHLVRQA
PGQGLEWMGWIHPTSGGTTYAQKFQGRWMTRDTSISTAYMELSRLTSDDTAVYYCARM
SQNYDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSGAPGQRVTISC
TGSSSNIGAGYDVNWYQQFPGTAPKIIVYGDRPSGAPDRFSGSKSGTSASLAITGLRAEDE
ADYYCQSWDSRLSSYVFGTGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 209)
>HER076_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggc agaggtgaaaaagcccggagagtctctgaagatctcctgtaagggctctggatacacctttaccaaccactggatcgcctgggt gcgccagatgcccgggaaaggcctggagtggatgggcatcatctatcctggtgactctgaaacgaggtacagcccgtccttcca aggccacgtcaccatctcagccgacaagtccatcagtaccgcctatttgcagtggagcaccctgaaggactcggactccgccat gtacttctgtgtgagacaggcccgtggctgggacgacggacgggctggatattattattccggtatggacgcctggggccaggga accctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtggtgct ccaggagccatcgttctcagtgtcccctggagggacagtcacactcacctgtggcttgcgctctgggtcagtctctactagtcacta ccccagctggtaccagcagaccccaggccaggctccacgcacgctcatttacagcacaaacactcgctcttctggggtccctga tcgcttctctggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctaattattactgtat gctatacatgggcagtggcatgtatgtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttct gacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaag gacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaa ctggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggt cagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcc cccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatga gctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatg ggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtgg acaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctgtctccgggtaaatga (SEQ ID NO: 210)
>HER076_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCKGSGYTFTNHWIAWVRQMP
GKGLEWMGIIYPGDSETRYSPSFQGHVTISADKSISTAYLQWSTLKDSDSAMYFCVRQARG
WDDGRAGYYYSGMDAWGQGTLVTVSSGGGGSGGGGSGGGGSAQAWLQEPSFSVSPG
GTVTLTCGLRSGSVSTSHYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTI
TGAQADDESNYYCMLYMGSGMYVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 211 )
>HER077_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctggggg aggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccg ccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaaggg ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagagatctgggaatagaccccctttggagtggttattacacaccccttgactattggggccgagggacaatggtc accgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacacgttatactgactcaaccgcc ctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaattccgttagctggt accagcagctcccaggaacggcccccaaactcctcatgtatactaacaatcagcggccctcaggggtccctgaccgattctctg gctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcgacatgggat gccagcctgaatacttgggtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaa actcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacacc ctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtac gtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtc ctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcg agaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgacc aagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagc cggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctc cctgtctccgggtaaatga SEQ ID NO: 212)
>HER077_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDL
GIDPLWSGYYTPLDYWGRGTMVTVSSGGGGSGGGGSGGGGSAHVILTQPPSASGTPGQ
RVTISCSGSSSNIGSNSVSWYQQLPGTAPKLLMYTNNQRPSGVPDRFSGSKSGTSASLAIS
GLQSEDEADYYCATWDASLNTWVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 213)
>HER078_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtccagctggtgcagtctggagc tgaggtgaaggagcctggggcctcagtgaaggtctcctgcaaggcctctggttacgacttttccaactatggtttcagctgggtgcg ccaggcccctggacaaggtcttgagtggatgggatggatcagctcttataatggttacacaaactatgcacagagactccaggg cagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacagctgt ctattactgtgcgagagatcgaggacttggaaactggtacttcgatctctggggccaaggcaccctggtcaccgtctcgagtggtg gaggcggttcaggcggaggtggcagcggcggtggcggatcgcagtctgtgctgactcagcctgcctccgtgtctgggtctcctgg acagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacaacacccaggc aaagcccccaaactcatgatttatgagggcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacac ggcctccctgacaatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaaccaggagcactcgagttt tcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac tggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 214)
>HER078_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKEPGASVKVSCKASGYDFSNYGFSWVRQA
PGQGLEWMGWISSYNGYTNYAQRLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARD
RGLGNWYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSITISCT
GTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAE
DEADYYCSSYTTRSTRVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 215)
>HER079_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcagatgcagctggtgcagtctggggg aggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatggcatgcactgggtcc gccaggctccaggcaaggggctggagtgggtggcagttatatcatatgatggaagtattaaatactatgcagactccgtgaagg gccgattcaccatctccagagacaattccaagaacacactgtatctacaaatgaacagcctgagagccgaggacacgggcgtt tattactgttcgaaagatcgctatagcagtggctggtacagctccgatgcttttgatatttggggccgagggacaatggtcaccgtct cgagtggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgtctgagctgactcaggaccctgctgtgtctgtgg ccttgggacagacagtcaggatcacatgccaaggagacagcctcagaagctattatgcaagctggtaccagcagaagccag gacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaa cacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtcattcccgggacagcagtggtaaccat gtgcttttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccca ccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgga cccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcca aagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagc ctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactaca agaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatg a (SEQ ID NO: 216)
>HER079_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQMQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQ
APGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTGVYYCSKDR
YSSGWYSSDAFDIWGRGTMVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRIT
CQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAED
EADYYCHSRDSSGNHVLFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 217)
>HER080_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggg aggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatggcatgcactgggtcc gccaggctccaggcaaggggctggagtgggtggcagttatatcatatgatggaagtattaaatactatgcagactccgtgaagg gccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgt attactgtgcgcgaactggtgaatatagtggctacgatacgagtggttacagcaattggggccaaggcaccctggtcaccgtctc gagtggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgcagtctgtgctgactcagccaccctcagcgtctg ggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcgggagtaacactgtaaactggtaccagcga ctcccaggagcggccccccaactcctcatctacaataatgaccagcggccctcagggatccctgaccgattctctggctccaagt ctggcacctcaggctccctggtcatcagtgggctccagtctgaagatgaggctgattactactgtgcgtcatgggatgacagtctga atggtcgggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacat gcccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcg tggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcct gcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccat ctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccagg tcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggca gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaatga (SEQ ID NO: 218)
>HER080_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQ
APGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTG
EYSGYDTSGYSNWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTI
SCSGSSSNIGSNTVNWYQRLPGAAPQLLIYNNDQRPSGIPDRFSGSKSGTSGSLVISGLQS EDEADYYCASWDDSLNGRVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 219)
>HER081_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtgcagtctggggg aggcttggtccagccgggggggtccctgagactctcctgtgcagcctctggattcacgtttagtacctatgccatgagttgggcccg ccaggctccagggaaggggctggagtgggtctcaagtattagtggtgatggtggaagaattctcgatgcagactccgcgaagg gccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacggcctgagagtcgaggacacggcccttt attactgtgcgagagcggacggtaactactggggcagggggacaatggtcaccgtctcttcaggtggaggcggttcaggcgga ggtggcagcggcggtggcggatcgcagtctgtgctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctc ctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcat gatttatgagggcagtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctc tgggctccaggctgaggacgaggctgattattactgcagctcatatacaaccaggagcactcgagttttcggcggagggaccaa gctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcct gggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg gtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagc cgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttc tatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggac tccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 220)
>HER081_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGGGLVQPGGSLRLSCAASGFTFSTYAMSWARQA
PGKGLEWVSSISGDGGRILDADSAKGRFTISRDNSKNTLYLQMNGLRVEDTALYYCARADG
NYWGRGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSITISCTGTSSDVG
GYNYVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC
SSYTTRSTRVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK (SEQ ID NO: 221 )
>HER082_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtcggggg gaggcgtggtccagcctggggggtccctgagactctcctgtgcagcgtctggattcaccttcagtggctatggcatgcactgggtc cgccaggctccaggcaaggggctggagtgggtggcatctgtacggaacgatggaagtaatacatactacacagactccgtga aggaccgattcaccatctccagagacaacaccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacgg ccgtatattactgtgccaagtcgagaagagtgatgtatggcacctcctattactttgactactggggcagaggcaccctggtcaccg tctcctcaggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgtctgagctgactcaggaccctgctgtgtctgt ggccttgggacagacagtcaggatcacatgccaaggagacagcctcagaagctattatgcaagctggtaccagcagaagcc aggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcagga aacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaac catgtggtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgc ccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg gaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga (SEQ ID NO: 222)
>HER082_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLQESGGGWQPGGSLRLSCAASGFTFSGYGMHWVRQ
APGKGLEWVASVRNDGSNTYYTDSVKDRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAKS
RRVMYGTSYYFDYWGRGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRIT
CQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAED
EADYYCNSRDSSGNHWFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 223)
>HER083_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtcgggcg caggactggtgaagccttcggggaccctgtccctcacctgcgctgtctctggtggctccatcagcagtggtaactggtggagttgg gtccgccagcccccagggaaggggctggagtggattggggaaatctctcatagtgggagcaccaactacaacccgtccctca agagtcgagtcaccatatcagtagacaagtccaagaaccagttctccctgaacctgagttctgtgaccgccgcagacacggcc gtgtattactgtgcgagagtaaggggtacggtgggggatacacggggacctgactactggggccagggaaccctggtcaccgt ctcgagtggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgtctgagctgactcaggaccctgctgtgtctgt ggccttgggacagacagtcaggatcacatgccaaggagacagcctcagaagctattatgcaagctggtaccagcagaagcc aggacaggcccctgtacttgtcatctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctcagga aacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaac catgtggtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgc ccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc ggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtg gaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagc aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga (SEQ ID NO: 224)
>HER083_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLQESGAGLVKPSGTLSLTCAVSGGSISSGNWWSWVRQ
PPGKGLEWIGEISHSGSTNYNPSLKSRVTISVDKSKNQFSLNLSSVTAADTAVYYCARVRGT
VGDTRGPDYWGQGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRITCQG
DSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADY
YCNSRDSSGNHWFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK (SEQ ID NO: 225)
>HER084_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggg aggcctggtcaagcctggggggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatgggatgcactgggtcc gccaggctccaggcaaggggctggagtgggtggcaggtattttttatgatggaggtaataaatactatgcagactccgtgaaggg ccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgta ttactgtgcgagagataggggctactactacatggacgtctggggcaaagggaccacggtcaccgtctcctcaggtggaggcg gttcaggcggaggtggctctggcggtggcggatcgcagtctgtgttgacgcagccgccctcagtgtctggggccccaggacaga gggtcaccatctcctgcactgggagaagctccaacatcggggcgggtcatgatgtacactggtaccagcaacttccaggaaca gcccccaaactcctcatctatggtgacagcaatcggccctcaggggtccctgaccgattctctggctccaggtctggcacctcagc ctccctggccatcactgggctccaggctgaagatgaggctgattattactgccagtcctatgacagcagcctgaggggttcggtatt cggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac tggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcca aagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 226)
>HER084_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQA
PGKGLEWVAGIFYDGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR
GYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISCTGR
SSNIGAGHDVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSGSRSGTSASLAITGLQAEDEA
DYYCQSYDSSLRGSVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK (SEQ ID NO: 227)
>HER085_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctggggg aggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccg ccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaaggg ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagaggcgggagtgggagtgactactggggccaggggacaatggtcaccgtctcgagtggaggcggcggttc aggcggaggtggctctggcggtggcggaagtgcacttaattttatgctgactcagccccactctgtgtcggggtctccggggaag acggtaaccatctcctgcacccgcagcagtggctacattgacagcaagtatgtgcagtggtaccagcagcgcccgggcagtgc ccccaccactgtgatctatgaggataaccgaagaccctctggggtccctgatcggttctctggctccatcgacagctcctccaactc tgcctccctcaccatctctggactggagactgaggacgaggctgactattactgtcagtcttatgatgacaccaatgtggtgttcggc ggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag cacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 228)
>HER085_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGG
SGSDYWGQGTMVTVSSGGGGSGGGGSGGGGSALNFMLTQPHSVSGSPGKTVTISCTRS
SGYIDSKYVQWYQQRPGSAPTTVIYEDNRRPSGVPDRFSGSIDSSSNSASLTISGLETEDE ADYYCQSYDDTNWFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 229)
>HER086_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtggggtgcagctggtggagtctgggg gaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctataacatgaactgggtc cgccaggctccagggaagggactggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgacg ggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccg tatattactgtgcgaaagataccagtggctggtacggggacggtatggacgtctggggccggggaaccctggtcaccgtctcga gtggtggaggcggttcaggcggaggtggcagcggcggtggcggatcggacatccagatgacccagtctccttccaccctgtctg catctattggagacagagtcaccatcacctgccgggccagtgagggtatttatcactggttggcctggtatcagcagaagccagg gaaagcccctaaactcctgatctataaggcctctagtttagccagtggggccccatcaaggttcagcggcagtggatcagggac agatttcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacaatatagtaattatccgctcactttcgg cggagggaccaagctggagatcaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccca gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaag ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct ggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 230)
>HER086_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGGVQLVESGGGLVKPGGSLRLSCAASGFTFSSYNMNWVRQA
PGKGLEWVSAISGSGGSTYYADSVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDTS
GWYGDGMDVWGRGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITC
RASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFAT
YYCQQYSNYPLTFGGGTKLEIKRDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK (SEQ ID NO: 231 )
>HER087_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctggggg aggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccg ccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaaggg ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgt attactgtgcgagagtcagcgggagccactttccattctttgactcctggggccaggggacaatggtcaccgtctcgagtggaggc ggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgactcagccaccctcggtgtcagtggcccca ggacagacggccagaattacctgtgggggagacaagattggacataaaagtgtgcattggtatcagcagaagccaggccag gcccctgtgttgctcgtctatgatgataggaagcggccctcagggatccctgagcgattctctggctccaactctgggaacacggc caccctgaccatcagcagggtcgaggccggggatgaggctgcctatcactgtcaggtgtgggatagaagtagtgacccttatgt cttcggaactgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtg cccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccct gaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgca taatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccagga ctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcc aaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac ctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacc acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaa cgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 232)
>HER087_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVS
GSHFPFFDSWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSVAPGQTARITC
GGDKIGHKSVHWYQQKPGQAPVLLVYDDRKRPSGIPERFSGSNSGNTATLTISRVEAGDE
AAYHCQVWDRSSDPYVFGTGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 233)
>HER_SMIPs_huVk3J_eader_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggt (SEQ ID NO: 234)
>HER_SMIPs_huVk3_Leader_Protein MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 235)
>HER_SMIPs_G4Sx3_Linker_CDS ggaggcggcggttcaggcggaggtggctctggcggtggcggaagt (SEQ ID NO: 236)
>HER_SMIPs_G4Sx3_Linker_Protein GGGGSGGGGSGGGGS (SEQ ID NO: 237)
>HER_SMIPs_SCCP_Hinge_CDS gagcccaaatcttctgacaaaactcacacatgcccaccgtgccca (SEQ ID NO: 238)
>HER_SMIPs_SCCP_Hinge_Protein EPKSSDKTHTCPPCP (SEQ ID NO: 239)
>HER_SMIP_Fc-Stop_CDS gacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcagt cttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccac gaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggt ctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtaca ccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatc gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttct tcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgc acaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 240)
>HER_SMIP_Fc_Stop_Protein
DVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 241 ) Her2_SlR3Bl_DP47_3A2
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROTPGKGLEWVSAISGSGGSTYYAN SVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARGGGYNPFDSWGOGTMVTVSS (SEQ ID NO: 251)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGAC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAAACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCGGGG GGGAGGCTACAACCCTTTTGACTCCTGGGGCCAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 252)
VL with CDR1 , CDR2 and CDR3 underlined
OSALTOP ASVSGSPGOSITISCTGTGSDVGGYNYVSWYOOHPGKAPKLMIYEVINRPSGISNR FSGSKSGNTASLTISGLOAEDEADYYCGSYSSSSTLVFGGGTKLTVL (SEQ ID NO: 253)
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCT
CCTGCACTGGAACCGGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGC
ACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTCATTAATCGGCCCTCAGGGATTT
CTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCC
AGGCTGAGGACGAGGCTGATTATTACTGCGGCTCATATTCAAGCAGCAGCACTCTTGTAT
TCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ LD NO: 254)
Her2_S 1R3 A1_DP47_11B7
VN with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAGAADYSNYFDFWGOGTMVTVSS (SEQ ID NO: 255)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGGGAGC
GGCGGACTACAGTAATTACTTTGACTTTTGGGGCCAAGGGACAATGGTCACCGTCTCGAG
T (SEQ DD NO: 256)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSGAPGORVTISCTGSSSNIGAGYD VHWYOOLPGAAPKLLIYGNINRPSGVPDR FSGSKSGTSASLAΓΓGLOAEDEGDYYCOSYDRSLSAKLFGGGTKVTVL (SEQ ID NO: 257)
CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCA
ACTTCCAGGAGCAGCCCCCAAACTCCTCATCTATGGGAACATCAATCGGCCCTCAGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCT
CCAGGCTGAGGATGAGGGTGATTATTACTGCCAGTCCTATGACAGAAGCCTGAGTGCTA
AGCIGTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ DD NO: 258) Her2_SlR3Al_DP47_llDl
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNALYLOMNSLRAEDTAVYYCARDLGIDPLWSGYYTPLDYWGRGTM VTVSS (SEQ ED NO: 259)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACGCGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA TCTGGGAATAGACCCCCTTTGGAGTGGTTATTACACACCCCTTGACTATTGGGGCCGGGG GACAATGGTCACCGTCTCGAGT (SEQ ID NO: 260)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODP ALSVALGOTVRITCOGDSLGGFHASWYOEKPGO AP VFVLYGKNNRPSGIPDRFS GSTSGNT AALTITGAQ AEDEAD YYCSSRDRSGNHRVFGGGTKLTVL (SEQ ID NO: 261 )
TCTTCTGAGCTGACTCAGGACCCTGCTCTGTCGGTGGCCTTGGGACAGACAGTCAGGATC
ACATGTCAAGGGGACAGCCTCGGAGGCTTTCATGCAAGCTGGTACCAGGAGAAGCCAGG
ACAGGCCCCTGTATTTGTCCTCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCG
ATTCTCTGGCTCCACCTCAGGTAACACAGCTGCCCTGACCATCACTGGGGCTCAGGCGGA
AGATGAGGCTGACTATTACTGTAGCTCCCGGGACAGAAGTGGTAACCATCGCGTCTTCGG
CGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 262)
Her2_SlR3Al_DP47_7F3
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDRALVGATRTFGYWGOGTTVTVSS (SEQ ID NO: 263)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA TCGAGCCCTAGTGGGAGCTACTCGAACTTTTGGCTACTGGGGGCAGGGGACCACGGTCA CCGTCTCGAGT (SEQ ED NO: 264)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPssvsGAPGORVTiscTGsssNiGAGYD VHWYOOLPGTAPKLLΓYGDTNRPSGVPDR
FSGSKSGTSASLAΓΓGLOAEDEADYYCOSFDSSLSGSVFGGGTKLTVL (SEQ ED NO: 265)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCA
GCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTGACACCAATCGGCCCTCAGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCT
CCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTTTGACAGCAGCCTCAGTGGTTC
GGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 266) Her2_SlR2B_DP47_4E3
VH with CDR1 , CDR2 and CDR3 underlined
EVLLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYAD SAKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDRVSNWNYΎGODSYFDYWGOGTM VTVSS (SEQ ID NO: 267)
GAGGTGCTGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATCAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGCGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGGA
CAGGGTCTCTAACTGGAACTACTACGGCCAGGACAGCTACTTTGACTACTGGGGCCAAG
GGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 268)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPPSASGTPGORVTISCSGSSSNIGSNYVYWYOOLPGTAPKVLIYRNNORPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCASWDGSLSGPVFGGGTKLTVL (SEQ ID NO: 269)
CAGGCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCAT
CTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAACT
CCCAGGAACGGCCCCCAAAGTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCC
CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCGTCCCTGGCCATCAGTGGGCTCC
GGTCCGAGGATGAGGCTGATTATTACTGTGCATCATGGGATGGCAGCCTGAGTGGTCCG
GTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 270)
Her2_SlR3Cl_DP47_2G2
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDRVSNWNYYGODSYFGYWGOGT MVTVSS (SEQ ID NO: 271)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGGA
CAGGGTCTCTAACTGGAACTACTACGGCCAGGACAGCTACTTTGGCTACTGGGGCCAGG
GGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 272)
VL with CDR1 , CDR2 and CDR3 underlined
SYELTOPPSASGTPGORVTISCSGSSSNIGSNTVTWYOOLPGTAPOLLFHNNDORPSGVPDRFS GSKSGTSGSLAISGLOSEDEADYYCSAWDDGLNAVIFGGGTKLTVL (SEQ ID NO: 273)
TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAACCTGGTACCAGCAGCTC CCAGGAACGGCCCCCCAACTCCTCTTCCATAATAATGACCAGCGGCCCTCAGGGGTCCCT GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGCTCCCTGGCCATCAGTGGGCTGCAG TCTGAGGATGAGGCTGATTATTACTGTTCAGCATGGGATGACGGCCTGAATGCTGTAATA TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 274) Her2_SlR3Al_DP47_llH6
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDRVYDFWSGYYTRYNWFDPWGRG TTVTVSS (SEQ ID NO: 275)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA TCGCGTTTACGATTTTTGGAGTGGTTATTATACGAGGTACAACTGGTTCGACCCCTGGGG GCGAGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO: 276)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPSSASGTPGORVTISCSGSSSNIGSNYVYWYOOLPGTAPKLLIYRNNORPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSSPVFGGGTKVTVL (SEQ ID NO: 277)
CAGGCTGTGCTGACTCAGCCGTCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC
TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTC
CCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCANGGGTCCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGG
TCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTAGTCCGGT
GTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 278)
Her2 S1R3A1_BMV_3B1
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGGGLVOPGGSLRLSCAASGFTFSSYPMHWVROAPGKGLEWVAWSFDGSKKYS ADSVKGRFTISRDISKNTLYLOMNSLRAEDTAVYYCAKDRYDSGTFYYGMDVWGRGTLVT VSS (SEQ ID NO: 279)
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGATCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATCCCATGCACTGGGTCCGCCAGGC
TCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTGTATCGTTCGATGGATCTAAGAAATACT
CTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACATCTCCAAGAACACGCTGT
ATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAAAGAT
CGCTATGATTCGGGGACTTTCTACTACGGCATGGACGTCTGGGGCCGGGGCACCCTGGTC
ACCGTCTCGAGT (SEQ ID NO: 280)
VL with CDR1 , CDR2 and CDR3 underlined
OSALTOPASVSGSRGOSRΠSCTGTTGDVGGYD YVSWYOOHPGRAPKLLIYGNSNRPSGVPD RFSASKSGNTASLTISGLOAEDEADYFCSTYAPPGΠMFGGGTKLTVL (SEQ ID NO: 28 I )
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGATCTCGTGGACAGTCGATCACCATC
TCCTGCACTGGAACCACTGGTGACGTTGGTGGTTATGACTATGTCTCCTGGTACCAACAG
CACCCAGGCAGAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGT
CCCTGATCGCTTCTCTGCCTCCAAGTCCGGCAATACGGCCTCCCTGACCATCTCTGGACTC
CAGGCTGAGGATGAGGCTGATTATTTCTGCAGCACATATGCACCCCCCGGTATTATTATG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 282) Her2_SlR3Al_DP47_6B9
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAISWVROAPGKGLEWVSAISGSGGSTYYAD SVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDSSRVGAYLVFDYWGRGTMVTVSS (SEQ ED NO: 283)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATAAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA
TTCGAGTAGGGTGGGAGCTTATCTGGTGTTTGACTACTGGGGCCGGGGGACAATGGTCAC
CGTCTCGAGT (SEQ ID NO: 284)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSGAPGORVTISCTGSSSNIGAGYD VHWYOOLPGTAPKLLIYGNSKRPSGVPDR FSGSKSGTSASLALTGLOAEDEADYYCOSYDSSLSGYVFGTGTKVTVL (SEQ ID
NO: 285)
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCAT
CTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGC
AGCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAAACGCCCCTCAGGG
GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCCTCACTGGG
CTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGT
TATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 286)
Her2_SlR2A_CS_10B8
VH with CDR1 , CDR2 and CDR3 underlined
OMOLVOSGAEVKKPGASVKVSCKASGYTFTSYYIHWVROAPGOGPEWMGIILPSGGSTSYA OEFOGRLSMTRDTSTSTVYMELSDLRSDDTAIYYCARDYDRSAYLDIWGRGTMVTVSS (SEQ ID NO: 287)
CAGATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT
TTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATACACTGGGTGCGACAGGC
CCCTGGACAGGGCCCTGAGTGGATGGGAATAATCCTCCCTAGTGGTGGCAGCACCAGCT
ACGCACAGGAGTTCCAGGGCAGACTCTCCATGACCAGGGACACGTCCACGAGCACAGTG
TACATGGAGCTGAGCGACCTGAGATCTGACGACACGGCCATTTATTATTGTGCGAGAGA
CTATGATAGGAGTGCTTATCTTGATATCTGGGGCCGAGGGACAATGGTCACCGTCTCGAG
T (SEQ ID NO: 288)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSASGTPGORVTISCSGSSSNIGRNTVNWYKOFPGTAPKLLΓYSDNKRPSGIPDRFS GSKSGTSASLAISGLOSGDEADYYCAAWDDSLNGHVVFGGGTKLTVL (SEQ DD NO: 289)
CAGTCTGTGTTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC
TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGAAATACTGTAAACTGGTACAAGCAGTTC
CCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATAAGCGGCCCTCAGGGATCCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAG
TCTGGGGATGAGGCTGATTATTACTGTGCCGCATGGGATGACAGCCTGAATGGCCATGTG
GTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 290) Her2_S 1R3 A1_DP47_7A6
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLQMNGLRVEDTAVYYCAKELVSRGSLTFDYWGKGTMVTVSS (SEQ DD NO: 291)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACGGCCTGAGAGTCGAGGACACGGCCGTGTATTACTGTGCGAAAGA ATTGGTCAGTAGAGGGAGCCTCACCTTTGACTACTGGGGCAAGGGGACAATGGTCACCG TCTCGAGT (SEQ ID NO: 292)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSGAPGOGVTISCTGSSSNIGADF AVHWYOOLPGTAPKLLINGSSHRPSGVPDR FSGSKSGPSASLAITGLOADDEADYFCOSYDYRLNALVFGGGTKLTVL (SEQ ID NO: 293)
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGGGGGTCACCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGATTTTGCTGTACACTGGTACCAACAA
CTTCCAGGGACAGCCCCCAAACTCCTCATCAATGGTAGCAGCCATCGGCCCTCAGGGGTC
CCTGACCGATTCTCTGGCTCCAAGTCTGGCCCCTCAGCCTCCCTGGCCATCACTGGGCTCC
AAGCCGACGATGAGGCTGATTATTTTTGCCAGTCCTATGACTACAGACTCAATGCTTTAG
IGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 294)
Her2_SlR3B2_DP47_2G3
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYΎCARGHKMGYFDYWGRGTLVTVSS
(SEQ ID NO: 295)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGG TCACAAAATGGGATACTTTGACTACTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 296)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODPAVSVALGOSVTΓΓCRGASLSNYYASWYOORPGOAPLLVVSDNNIRPSGIPDRFSG
SRSGTTASLSΓΓGAOAEDEADYYCHSRASSDTHVRVFGGGTKLTVL (SEQ ID NO: 297)
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCCGTGGCCTTGGGACAGTCAGTCACCATC
ACGTGTCGGGGAGCCAGCCTCAGCAACTATTATGCAAGCTGGTACCAGCAGAGGCCAGG
ACAAGCCCCTCTACTTGTCGTCTCTGATAACAACATCCGGCCCTCAGGGATCCCAGACCG
ATTCTCTGGCTCCAGGTCAGGAACCACAGCTTCCTTGAGCATCACTGGGGCTCAGGCGGA
AGATGAGGCTGACTATTACTGTCACTCCCGTGCCAGCAGTGACACCCATGTCCGGGTGTT
TGGCGGCGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 298) Her2_SlR2B_CS_6Hl 1
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKOPGESLKISCKGSGYSFSNYWIGWVROMPGKGLEWMGΠYPDDSDTRYSP SFOGOVTISADRSISTAYLOWSSLKASDTATYYCARGNVINGNTNAFDIWGRGTTVTVSS
(SEQ ID NO: 299)
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAACAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGATACAGCTTTAGCAACTACTGGATCGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGATGACTCTGATACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAGGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCACGTATTACTGTGCGAGAGGA
AATGTTATAAATGGAAATACCAATGCTTTTGATATCTGGGGGCGGGGGACCACGGTCAC
CGTCTCGAGT (SEQ ID NO: 300)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVIOEPSFSVSPGGTVTLTCGLSSGSVSTGYYPSWYOOTPGOAPRTLIYNTNSRSSGVPDR FSGSILGNKAALTITGAOADDESDYYCVLYMGSGISVFGGGTKLTVL (SEQ ID NO: 301)
CAGGCTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTACTGGTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAACACAAACAGTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGCTGTATATGGGTAGTGGCATTTCGGTA
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 302)
Her2_SlR3Al_DP47_10Gl
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAGGRVGSTAAFDTWGOGTMVTVSS (SEQ ID NO: 303)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGGGAGG TAGAGTGGGATCTACGGCGGCTTTTGATACATGGGGCCAGGGGACAATGGTCACCGTCT CGAGT (SEQ ID NO: 304)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSGAPGORV AISCTGSSSNIGAGYDVHWFOOLPGTAPKLLIFGNKNRPSGVPDR FSASKSGTAASLAΓΓGLOAEDEGDYYCOSYDSSLSGVIFGRGTKLTVL (SEQ ID NO: 305)
CAGTCTGTGTTAACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCGCCAT
ATCCTGTACGGGGAGCAGCTCCAATATTGGGGCAGGTTATGATGTACACTGGTTTCAGCA
ACTTCCAGGAACAGCCCCCAAACTCCTCATCTTTGGTAACAAGAATCGGCCCTCAGGGGT
CCCCGACCGATTCTCTGCCTCTAAGTCTGGCACCGCAGCCTCCCTGGCCATCACTGGGCT
CCAGGCTGAGGATGAGGGTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTGT
GATCTTCGGCAGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 306) Her2_SlR3Al_DP47_7Cl
VH with CDR1, CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYSAD SVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAGGRVGSTAAFDTWGRGTTVTVSS (SEQ ED NO: 307)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT CCGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGGGAGG TAGAGTGGGATCTACGGCGGCTTTTGATACATGGGGGCGAGGGACCACGGTCACCGTCT CGAGT (SEQ ED NO: 308)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPSSVSGAPGORVTISCTGSSSNIGAGYD VHWYOOLPGTAPKLLEYGNSNRPSGVPDR FSGSKSGTSASLAITGLOAEDEADYYCOSYDSSLRGYVFGTGTKLTVL (SEQ ED
NO: 309)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCA
GCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCT
CCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGGTTA
TGTCTTCGGAACTGGGACCAAGCTGACCGTCCTA (SEQ ED NO: 310)
Her2_SlR2A_DP47_5D6
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARGSRVGTIWGSLDDWGKGTMVTVSS (SEQ ED NO: 311)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGG
CAGTAGAGTGGGGACGATTTGGGGAAGCCTTGACGACTGGGGCAAAGGGACAATGGTCA
CCGTCTCGAGT (SEQ ED NO: 312)
VL with CDR1 , CDR2 and CDR3 underlined
ETTLTOSPGTLSLSPGERATLSCRASOSSSSSYLAWYOOKPGOAPRLLIYAASSRATGVPDRFS GSGSGTDFTLTISRLEPEDFAVYYCOOYGSSRFT FGQGTRLEEKR (SEQ ED NO: 313)
GAAACGACACTCACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC
CCTCTCCTGCAGGGCCAGTCAGAGTAGTAGCAGCAGCTACTTAGCCTGGTACCAGCAGA
AACCTGGCCAGGCTCCCAGGCTCCTCATCTATGCTGCATCCAGCAGGGCCACTGGCGTCC
CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTG
GAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACGGTTCACCTTC
GGCCAAGGGACACGACTGGAGATTAAACGT (SEQ ED NO: 314) Her2_SlR3Al DP47_11F6
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCGRGSRVGTIWGSLDFWGOGTMVTVSS (SEQ ID NO: 315)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGGGAGAGG
CAGCAGAGTGGGGACGATTTGGGGAAGCCTTGACTTTTGGGGCCAAGGGACAATGGTCA
CCGTCTCGAGT (SEQ ID NO: 316)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSAAPGORVTISCSGKSSNIGGNSVAWYOOLPGTAPKVLIYDNDKRPSGVPDRF SGSKSGTSATLGITGLOTGDEADYYCGSWDSSLGVGMFGGGTKVTVL (SEQ ID NO: 317)
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCGGGACAGAGGGTCACCATC
TCCTGCTCTGGAAAGAGCTCCAACATTGGCGGTAATTCTGTGGCCTGGTACCAGCAACTC
CCGGGAACAGCCCCCAAAGTCCTCATTTATGACAATGATAAGCGACCCTCAGGGGTTCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTGCAG
ACTGGGGACGAGGCCGATTATTACTGCGGATCCTGGGATAGCAGCCTGGGTGTTGGGAT
GTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO : 318)
Her2_SlR3Al_DP47_l 1D3
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARGYSGSSFDAWGOGTMVTVSS (SEQ ID NO: 319)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGGG CTACAGTGGAAGTTCCTTTGACGCCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 320)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSASGTPGORVTISCSGSSSNIGSKSVYWYOOLPGAAPKLLIYRNSORPSGVPDRFS ASKSGTSASLAISGLRSEDEADYYCAAWDGSLSGHFFGTGTKLTVL (SEQ ID NO: 321 )
CAGTCTGTGTTGACGCAGCCGCCATCAGCGTCCGGGACCCCCGGGCAGAGGGTCACCAT
CTCTTGTTCTGGAAGCAGCTCCAATATCGGAAGTAAGTCTGTATACTGGTACCAGCAACT
CCCAGGAGCGGCCCCCAAACTCCTCATCTACAGGAATAGTCAGCGGCCCTCAGGGGTCC
CTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCTGCCTCCCTGGCCATCAGTGGGCTCCG
GTCCGAGGATGAGGCTGACTATTACTGTGCAGCATGGGATGGCAGCCTGAGTGGACATT
ICTTCGGAACTGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 322) Her2_SlR3Al_CS_8A8
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKPGESLKISCKGSGYSFTSYWIGWVROMPGKGLEWMGΠYPGDSDTRYSP SFOGOVTISADKSISTAYLOWSSLKASDTAMFYCARLNDSSGYTTNFDYWGOGTLVTVSS
(SEQ ID NO: 323)
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGCTCTGGATACAGCTTTACCAGCTATTGGATCGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTTTTACTGTGCGAGACTCA
ATGATAGTAGTGGTTATACGACTAACTTTGACTACTGGGGCCAAGGCACCCTGGTCACCG
TCTCGAGT (SEQ ID NO: 324)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVIOEPSFSVSPGGTVTLTCGLSSGSVSTRYNPSWYOOTPGOAPRTLIYSTNTRSSGVPDRF SGSILGNKAALTITGAOADDESDYYCALYMGSGIWVFGGGTKLTVL (SEQ ID NO: 325)
CAGGCTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTACTCGTTACAACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGTACAAACACTCGTTCTTCTGGGGTC
CCTGACCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGCGCTGTATATGGGTAGTGGCATTTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 326)
Her2_SlR3Al_BMV_5D10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVESGGGLVOPGGSLRLSCAASGFTFDSYAMSWVROAPGKGLEWVSIISGRDGYTYYT DSVKGRFTISRDNSKNTVYLOMNSLRAEDTGVYYCARNGEWPGILDYWGRGTMVTVSS (SEQ ID NO: 327)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCTGCCTCTGGATTCACCTTTGACAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTAGTGGTAGAGATGGTTACACATACT
ACACAGACTCCGTGAAGGGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGGTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGGTGTGTATTATTGTGCGAGAAA
TGGGGAGTGGCCCGGAATCTTAGACTACTGGGGCAGGGGGACAATGGTCACCGTCTCCT
CA (SEQ DD NO: 328)
VL with CDR1 , CDR2 and CDR3 underlined
DIOMTOSPSTLSASIGDRVTITCRASEGΠΉWLAWYOOKPGKAPKLLIYKASSLASGAPSRFS GSGSGTDFTLTISSLOPDDFATYYCOOYSNYPLTFGGGTKLEIKR (SEQ ID NO: 329)
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACC
ATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCA
GGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCA
AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGA
GGGACCAAGCTGGAGATCAAACGT (SEQ ID NO: 330) Her2_SlR3Al_DP47_llCl
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAASNSYYYFDYWGOGTLVTVSS (SEQ ID NO: 331)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGGCGAG TAATAGTTATTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 332)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODP AVSV ALGOTVRITCOGDSLRNFYPSWYOOKPGOAPVLVIYGKNIRPSGIPDRFSG SGSGSTASLTITGAOAEDEAD YYCNSRDSSGKHMGWFGGGTKLTVL (SEQ ID NO: 333)
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATC ACATGCCAAGGAGACAGCCTCAGAAACTTTTATCCAAGTTGGTATCAGCAGAAGCCAGG ACAGGCCCCTGTTCTTGTCATTTATGGTAAAAATATTCGGCCCTCAGGGATCCCAGACCG ATTCTCTGGCTCCGGCTCAGGAAGCACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGA AGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAAACATATGGGGGTGG TATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 334)
Her2_SlR3Al_DP47_4El
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDMAVYYCARTPGYSSGWYSVWGRGTLVTVSS (SEQ ID NO: 335)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACATGGCCGTGTATTACTGTGCGAGGAC
TCCCGGGTATAGCAGTGGCTGGTACTCGGTTTGGGGCCGGGGCACCCTGGTCACCGTCTC
GAGT (SEQ DD NO: 336)
VL with CDR1 , CDR2 and CDR3 underlined
OSVVTOPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOVPGTAPKLLIYGNNNRPSGVPD RFSGSKSGTSASLAΓΓGLOPEDEVDYYCOSYDRSLSGYIFGSGTKVTVL (SEQ ID NO: 337)
CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCAT
CTCCTGTACTGGGAGCAGCTCCAACATCGGGGCAGGGTATGATGTTCACTGGTACCAGCA
GGTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCCTCGGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCT
CCAGCCTGAGGATGAAGTTGATTATTACTGCCAGTCCTATGACCGCAGCCTGAGTGGTTA
TATCTTCGGAAGTGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 338) Her2_SlR3Al_DP47_10El
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARVGSFGDYKDKSGYGFYFDYWGOG TLVTVSS (SEQ ID NO: 339)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGT TGGGTCGTTTGGTGATTACAAAGATAAAAGTGGTTACGGCTTCTACTTTGACTACTGGGG CCAAGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 340)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSASGTLGOTVFISCSGSSSNIGSNSVSWYOOLPGTAPKFLIYHNNORPSGVPERFS GSKSGTSASLAIRGLOSEDEADYYCASWEDSLNGWVFGGGTKLTVL (SEQ ID NO: 341)
CAGTCTGTGCTGACTCAGCCACCCTCGGCGTCTGGGACCCTCGGGCAGACGGTCTTCATC
TCTTGTTCTGGAAGCAGTTCCAACATCGGAAGTAATTCTGTGAGTTGGTACCAGCAGCTC
CCAGGAACGGCCCCCAAATTTCTCATTTATCATAATAATCAGCGGCCCTCAGGGGTCCCT
GAGCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCCGTGGGCTCCAG
TCTGAGGATGAGGCTGATTACTACTGTGCATCTTGGGAGGACAGCCTGAATGGTTGGGTG
TTCGGCGGGGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 342)
Her2_SlR3Al_CS_llC3
VH with CDR1 , CDR2 and CDR3 underlined
OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYGISWVROAPGOGLEWMGWISAYNGNTNY AOKLOGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGSGYCSGGSCYVGWFDPWGRG TMVTVSS (SEQ ID NO: 343)
CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT
CTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACT
ATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCC
TACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGI
GGGGTCGGGATATTGTAGTGGTGGTAGCTGCTACGTGGGCTGGTTCGACCCCTGGGGCCG
GGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 344)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODP AVSV ALGOTVKITCOGDSLSAYYATWYOOKPGO AP VLVIYGKNKRPSGIPDRFS GSKSGNTASLTITGAOAEDEADYYCNSRDSSGNDHYVFGTGTKLTVL (SEQ ID NO: 345)
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAAGATC
ACATGCCAAGGAGACAGCCTCAGTGCCTATTATGCAACCTGGTACCAGCAGAAGCCAGG
CCAGGCCCCTGTACTTGTCATCTATGGTAAAAACAAGCGGCCGTCCGGGATCCCAGACCG
ATTCTCTGGCTCCAAGTCAGGAAACACAGCTTCCTTGACCATCACGGGGGCTCAGGCGGA
AGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAATGATCATTATGTCTT
CGGAACTGGGACCAAGCTGACCGTTCTA (SEQ ID NO: 346) Her2_SlR3Al_CS_13Hll
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVRKPGASVKVSCRASGYNFKDYYLHWVRQAPGEGLEWMGWINPHAGTTK YAONFOHRΠMTRDTTITTAYMELSSLKSDDTAIYFCTRYYFDSSGYFRFDPWGOGTMVTVSS
(SEQ ID NO: 347)
GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTCAGTCAAGGT
CTCCTGCAGGGCTTCTGGATACAACTTCAAAGACTACTATTTGCACTGGGTGCGCCAGGC
CCCTGGAGAAGGGCTTGAGTGGATGGGGTGGATCAACCCTCACGCTGGTACCACAAAAT
ATGCACAGAATTTTCAGCACAGGATTATTATGACCAGGGACACGACCATCACCACAGCC
TACATGGAACTGAGCAGTCTGAAATCTGACGACACAGCCATTTATTTCTGTACCAGATAC
TACTTTGACAGTAGTGGTTATTTTAGGTTCGACCCCTGGGGCCAAGGGACAATGGTCACC
GTCTCGAGT (SEQ ID NO: 348)
VL with CDR1 , CDR2 and CDR3 underlined
OSVVTOPPSVSGAPGOKVTISCSGSSSNIGNNYVSWYOOLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLOTGDEADYYCGTWDSSLSAGVFGGGTKVTVL (SEQ ID NO: 349)
CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGACAGAAGGTCACCAT
CTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCT
CCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCC
TGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCA
GACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCTGGGG
TGTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 350)
Her2_S 1R3 A1_CS_2D9
VH with CDR1 , CDR2 and CDR3 underlined
OMOLVOSGAEVKKPGESLKMSCKGSGYSFTSYWIGWVROMPGKGLEWMGIIYPGNSDTRY NPSFEGOVTISADKSINTAFLOWNSLKASDTAIYYCARAPWVGAFDTWGOGTMVTVSS (SEQ ID NO: 351)
CAGATGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT GTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGAT GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTAACTCCGATACCAGAT ACAACCCGTCCTTCGAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAACACCGCCT TCCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATATATTATTGTGCGCGGGCTC CCTGGGTGGGTGCTTTTGATACTTGGGGCCAGGGGACAATGGTCACCGTCTCTTCA (SEQ ID NO: 352)
VL with CDR1 , CDR2 and CDR3 underlined
DIVMTOSPSTLSASVGDRVTITCRASOGISSWLAWYOOKPGRAPKVLIYKASTLESGVPSRFS GSGSGTDFTLTISSLOPEDFATYYCOOSYSTPWTFGOGTKLEIKR (SEQ ID NO: 353)
GACATCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACC
ATCACTTGCCGGGCCAGTCAGGGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCA
GGGAGAGCCCCTAAGGTCTTGATCTATAAGGCATCTACTTTAGAAAGTGGGGTCCCATCA
AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT
GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAA
GGGACCAAGCTGGAGATCAAACGT (SEQ ID NO: 354) Her2_S 1R2 A_CS_3D4
VH with CDR1 , CDR2 and CDR3 underlined
OMOLVOSGAEVTaCPGASVKVSCKSSGYTFKDYYINWVROAPGOGLEWVGWINPKNGITKY SONFOGRVSMTTDTSISTVYMDLRGLTSDDTAVYYCARDANRLRVGWFDPWGQGTLVTVS S (SEQ ID NO: 355)
CAGATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGGCCTCAGTGAAAGT
CTCCTGCAAGTCTTCTGGATACACCTTCAAGGACTACTATATCAACTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGGTGGGATGGATCAACCCTAAAAATGGTATCACAAAAT
ATTCGCAGAATTTTCAGGGCAGGGTCTCCATGACCACGGATACGTCCATCAGCACAGTCT
ACATGGACCTGAGAGGTCTGACATCTGACGACACGGCCGTTTATTATTGTGCGAGAGAC
GCGAACCGCCTTAGGGTGGGCTGGTTCGACCCCTGGGGCCAAGGAACCCTGGTCACCGT
CTCGAGT (SEQ ID NO: 356)
VL with CDR1 , CDR2 and CDR3 underlined
OA VLTOPSSVSGSPGORVsiscTGSSSNiGAGYD VHWYOOLPGTAPKLLIYGNΓNRPSGVPDR
FSGSKSGTSASLAITGLOAEDEADYYCOSYDSSLRAAVFGGGTKVTVL (SEQ ID NO: 357)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGTCCCCAGGGCAGAGGGTCAGCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACATTGGTATCAACAA
CTTCCAGGAACAGCCCCCAAACTCCTCATCTACGGTAACATCAATCGGCCCTCAGGGGTC
CCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTC
CAGGCTGAAGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGGGCTGC
GGTATTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 358)
Her2_SlR3Al_DP47_2H6
VN with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDFWFGLPPSFFDSWGOGTMVTVSS (SEQ ID NO: 359)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGA
TTTCTGGTTTGGACTACCACCTTCCTTCTTTGACTCTTGGGGCCAAGGGACAATGGTCACC
GTCTCGAGT (SEQ ID NO: 360)
VL with CDR1 , CDR2 and CDR3 underlined
QSVLTQPPSVSVSPGOKASITCSGERMGDKYAAWYOOKPGOSPILVIYODTKRPSGIPERFSG SNSGNTATLTISGTQDMDEADYYCOVWDSSTGVFGGGTKVTVL (SEQ ID NO: 361)
CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGAAGGCCAGCATC
ACCTGCTCTGGAGAAAGAATGGGGGATAAATATGCTGCCTGGTATCAGCAGAAGCCAGG
CCAGTCACCTATACTGGTCATCTATCAAGATACAAAGCGGCCCTCAGGGATCCCTGAGCG
ATTCTCTGGCTCCAACTCTGGGAACACAGCCACGTTGACCATCAGCGGGACCCAGGACAT
GGATGAGGCTGACTATTACTGTCAGGTGTGGGACAGCAGCACTGGGGTATTCGGCGGAG
GGACCAAGGTCACCGTCCTA (SEQ ID NO: 362) Her2 S1R3A1_DP47_4G1
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDLNPYSVVTLGMDVWGRGTMVTV SS (SEQ ED NO: 363)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGA
TCTGAACCCTTATTCAGTGGTAACTCTCGGTATGGACGTCTGGGGCAGAGGGACAATGGT
CACCGTCTCGAGT (SEQ ID NO: 364)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSV APGO AARIPCGGDNIGSKSVHWYOORPGOAPVLVVFDDSDRPSGIPERFSG SNSGHTATLTINRVEPGDEAEYYCEVWDGGERHVVFGGGTKLTVL (SEQ ID NO: 365)
CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGGCGGCCAGGATT
CCCTGTGGGGGAGACAACATTGGAAGTAAGAGTGTTCACTGGTACCAGCAGAGGCCAGG
CCAGGCCCCTGTCCTGGTCGTCTTTGATGATAGTGACCGGCCCTCAGGGATCCCTGAGCG
ATTCTCTGGCTCCAATTCTGGGCACACGGCCACCCTGACCATCAACAGGGTCGAACCCGG
GGATGAGGCCGAGTATTATTGTGAGGTGTGGGATGGTGGCGAGAGACATGTGGTATTCG
GCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 366)
Her2_SlR2A_DP47_3Cl
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDLTGPNHWFFDLWGRGTTVTVSS (SEQ ID NO: 367)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCCAGAGA
TTTAACTGGCCCAAACCACTGGTTCTTCGATCTCTGGGGGCGGGGGACCACGGTCACCGT
CTCGAGT (SEQ ID NO: 368)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSVSPGOTASISCSGHRLGDKYVSWYRORPGOSPVLVIYODEKRYSGISERFSGS NSGNVATLTITGTQAMDEADYHC OAWDSTTWFGGGTKLTVL (SEQ ID NO: 369)
CAGTCTGTGCTGACTCAGCCACCCTCCGTGTCCGTGTCCCCAGGACAGACAGCCAGCATC
TCCTGCTCTGGACATAGATTGGGCGATAAGTATGTTAGTTGGTATCGGCAGAGGCCGGGC
CAGTCCCCTGTGCTGGTCATCTATCAAGATGAGAAGAGGTACTCAGGGATCTCTGAGCGA
TTCTCTGGCTCCAACTCTGGGAACGTAGCCACTCTGACCATCACCGGGACCCAGGCTATG
GATGAGGCTGACTATCACTGTCAGGCGTGGGACAGCACCACTGTGGTGTTCGGCGGAGG
GACCAAGCTGACCGTCCTA (SEQ ID NO: 370) Her2_SlR3Al_DP47_7B2
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWYSAISGSGGSTYYA DSVKGRFriSRDNSKJSrrLYLOMNSLRAEDTAVYYCARDRRPRDDAFDMWGRGTTVTVSS (SEQ ID NO: 371)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA TAGACGGCCGAGGGATGATGCTTTTGATATGTGGGGGAGAGGGACCACGGTCACCGTCT CGAGT (SEQ ID NO: 372)
VL with CDR1 , CDR2 and CDR3 underlined
DIOMTOSPSSVSASVGDRVSITCRASOGIGSWLFWYOOKPGKAPILLMSAVSGLOSGVPSRFS GSGSGTDFTLTISSVOPEDFATYYCOOAHSFPITFGOGTRLEIKR (SEQ ID NO: 373)
GACATCCAGATGACCCAGTCTCCCTCTTCTGTGTCTGCTTCTGTTGGAGACAGAGTCAGC
ATCACTTGTCGGGCGAGTCAGGGAATTGGCAGCTGGTTATTCTGGTATCAGCAGAAACCA
GGGAAAGCCCCTATCCTCCTGATGTCCGCTGTGTCCGGTTTGCAAAGTGGAGTCCCATCA
CGATTCAGCGGCAGCGGATCTGGGACAGATTTCACTCTCACGATCAGCAGCGTACAGCCT
GAGGATTTTGCAACTTACTATTGTCAACAGGCTCACAGTTTCCCTATCACCTTCGGCCAA
GGGACACGACTGGAGATTAAACGT (SEQ DD NO: 374)
Her2_SlR3B2_DP47_4E2
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDRVSNWNYYGODSYFDYWGOGT MVTVSS (SEQ ID NO: 375)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGGA
CAGGGTCTCTAACTGGAACTACTACGGCCAGGACAGCTACTTTGACTACTGGGGCCAGG
GGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 376)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSASGAPGORVTISCSGTNSMGSNNVNWYOOLPGKAPRLLIYNNNORPSGVPDRF SGSKSGTSASLAISGLOSELEADYYCSAWDDSLHGPVFGGGTKLTVL (SEQ ID NO: 377)
CAGTCTGTGCTGACTCAGCCACCCTCCGCGTCTGGGGCCCCCGGGCAGAGGGTCACCATT
TCTTGTTCTGGGACCAACTCCAACATCGGAAGTAATAATGTAAACTGGTATCAGCAACTC
CCAGGAAAGGCCCCCAGACTCCTCATCTACAATAATAATCAGAGGCCCTCAGGGGTCCC
TGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCA
GTCTGAGCTTGAGGCTGATTATTATTGTTCAGCATGGGATGACAGCCTGCATGGTCCGGT
GTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 378) Her2_SlR3Al_CS_16C2
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVESGGGLAOPGGSLRLSCAASGLTFTTYAMSWVROAPGKGLEWVSSISGSGHSTYYA DSVKGRFTISRDISKNTLYLOMNSLRAEDTA VYYCAKDSSAFGFVHGAFDIWGOGTLVTVSS (SEQ ID NO: 379)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTAACCTTTACCACCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGGAAGTGGTCATAGCACATATT
ACGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACATTTCCAAGAACACGTTGT
ATCTGCAAATGAACAGCCTCAGAGCCGAGGACACGGCCGTCTATTACTGTGCGAAAGAT
TCGTCGGCTTTTGGGTTTGTACACGGTGCTTTTGATATCTGGGGCCAGGGAACCCTGGTC
ACCGTCTCGAGT (SEQ ID NO: 380)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODP AASVALGOTVSITCOGDSLRNYWASWYOOKPGOAPVLVIYGKNTRPSGIPDRFS GSTSGNTASLTITGAOAEDEADYYCNSRDSGHRLLFGGGTKLTVL (SEQ ID
NO: 381)
TCTTCTGAGCTGACTCAGGACCCTGCTGCGTCTGTGGCCTTGGGACAGACAGTCAGCATC ACATGCCAAGGAGACAGCCTCAGAAACTATTGGGCTAGCTGGTACCAGCAGAAGCCAGG ACAGGCCCCTGTACTTGTCATCTATGGTAAAAATACCCGGCCCTCAGGGATCCCAGACCG ATTCTCTGGCTCCACCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGA GGATGAGGCTGACTATTACTGCAACTCCCGGGACAGTGGTCACCGTCTTCTTTTCGGCGG AGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 382)
Her2_SlR3Al_CS_l 1E5
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKRPGESLKISCRASGYIFTNNWVAWVROOPGKGLEWMGΠYPGDSDTRYSP SFOGOVTFSADTSINTAYLOWNSLKASDTATYFCAREAYNSYEYYGMDVWGRGTTVTVSS
(SEQ ID NO: 383)
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAAGTCAAGAGGCCCGGAGAGTCTCTGAAGAT
CTCCTGTAGGGCCTCTGGATACATCTTTACGAACAATTGGGTCGCCTGGGTGCGCCAGCA
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGACACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACTTTCTCGGCCGACACGTCCATCAACACCGCCT
ACCTACAGTGGAATAGCCTGAAGGCCTCGGACACCGCCACTTACTTCTGTGCGCGAGAG
GCCTACAACTCATACGAATATTACGGTATGGACGTCTGGGGGCGAGGGACCACGGTCAC
CGTCTCGAGT (SEQ ID NO: 384)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVIOEPSFSVSPGGTVTLTCGLSSGSVSTNYYPSWYOOTPGO APRTLIYNTNTRSSGVPDRF SGSILGNKAALTITGAOADDESDYYCVLYMGSGISVFGGGTKVTVL (SEQ ID NO: 385)
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTACTAATTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAACACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGCTGTATATGGGTAGTGGCATTTCGGTG
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 386) Her2 S1R3A1 CS 16D7
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVESGAEVKKPGESLKJSCKASGYIFΓNNWIAWVRQOPGKGLEWMGΠYPGDSDTRYSP SFOGRVTFSADTSINTAYLOWSSLKASDTATYYCAREAYNSYEYYGMDVWGOGTMVTVSS
(SEQ ID NO: 387)
GAGGTGCAGCTGGTGGAGTCCGGAGCAGAAGTCAAAAAGCCCGGAGAGTCTCTGAAGAT
CTCCTGTAAGGCTTCTGGATACATCTTTACGAACAATTGGATCGCCTGGGTGCGGCAGCA
GCCCGGGAAAGGCCTGGAGTGGATGGGAATCATCTATCCTGGTGACTCTGACACCAGAT
ACAGCCCGTCCTTCCAGGGCCGGGTCACTTTCTCAGCCGACACGTCCATCAACACCGCCT
ACCTCCAGTGGAGTAGCCTGAAGGCCTCGGACACCGCCACTTACTACTGTGCGAGAGAG
GCCTACAACTCATACGAGTACTACGGTATGGACGTCTGGGGCCAAGGGACAATGGTCAC
CGTCTCGAGT (SEQ ID NO: 388)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVLOEP AFSVSPGGTVTLTCGLSSGSVSTSYYPSWYOOTPGOPPRTLIYNTNTRSSGVSDR FSGSILGNK-AALTITGAOAEDESDYYCVLYMGSGISVFGGGTKLTVL (SEQ ID NO: 389)
CAGACTGTGGTGCTCCAGGAGCCAGCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACCTGTGGCTTGAGCTCTGGCTCAGTCTCTACTAGTTACTACCCCAGTTGGTACCAGCAG
ACCCCAGGCCAGCCTCCACGCACGCTCATCTACAACACAAACACCCGCTCTTCTGGGGTC
TCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCCGAAGATGAATCTGATTATTACTGTGTTCTGTATATGGGTAGTGGCATTTCGGTA
TTCGGCGGGGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 390)
Her2_SlR2A_CS_10B10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKPGASVRVSCKGSGNTFTGHYIHWVROAPGOGLEWLGWIDPNTGDIOYS ENFKGSVTLTRDPSINSVFMDLIRLTSDDTAMYYCAREGAGLANYYYYGLDVWGRGTMVT VSS (SEQ ID NO: 391)
GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGGGT
CTCCTGCAAGGGTTCTGGAAACACCTTCACCGGCCACTACATCCACTGGGTGCGACAGGC
CCCTGGACAAGGACTTGAGTGGCTGGGATGGATCGACCCTAACACTGGTGACATACAGT
ATTCAGAAAACTTTAAGGGCTCGGTCACCTTGACCAGGGACCCATCCATCAACTCAGTCT
TCATGGACCTGATCAGGCTGACATCTGACGACACGGCCATGTATTACTGTGCGAGAGAA
GGTGCCGGGCTCGCCAACTACTATTACTACGGTCTGGACGTCTGGGGCCGAGGGACAAT
GGTCACCGTCTCGAGT (SEQ ID NO: 392)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVLOEPSFSVSPGGTVTLTCGLNFGSVSTAYYPSWYOOTPGOAPRTLIYGTNIRSSGVPDR FSGSIVGNKAALTITGAOTEDESDYYCALYMGSGMLFGGGTKVTVL (SEQ ID NO: 393)
CAGACTGTGGTGCTCCAGGAGCCTTCGTTCTCAGTGTCCCCTGGGGGGACAGTCACACTC
ACTTGTGGCTTGAACTTTGGCTCAGTCTCTACTGCTTACTACCCCAGTTGGTACCAGCAGA
CCCCAGGCCAAGCTCCACGCACGCTCATCTACGGCACAAATATTCGTTCCTCTGGGGTCC
CGGATCGCTTCTCTGGCTCCATCGTAGGGAACAAAGCTGCCCTCACCATCACGGGGGCCC
AGACAGAAGATGAGTCTGATTATTATTGTGCGCTGTATATGGGTAGTGGCATGCTCTTCG
GCGGCGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 394) Her2_SlR3Al_CS_15C2
VH with CDR1 , CDR2 and CDR3 underlined
OVOLOKSGPGLVKP AGTLSLTCAVSGDSISSNHWWNλVVROSPGKGLEWIGEIFHSDIRILNPS LKRRVSMSVDRSKDOFSLOLTSVTAADTAVYYCARGFHGDSGRGLDTWGRGTLVTVSS (SEQ ID NO: 395)
CAGGTGCAGCTGCAGGAGTCCGGCCCAGGACTCGTGAAGCCTGCGGGGACTCTGTCCCT
CACCTGCGCTGTCTCCGGTGACTCCATCAGCAGCAATCACTGGTGGAATTGGGTCCGCCA
GTCCCCAGGGAAGGGACTGGAATGGATTGGTGAAATCTTTCATAGTGACATTCGCATCCT
CAACCCGTCCCTCAAGAGGCGCGTCTCCATGTCAGTCGACAGGTCCAAGGACCAATTCTC
CCTGCAACTGACCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGTTT
CCATGGTGACTCCGGCAGAGGACTTGACACCTGGGGCAGAGGAACCCTGGTCACCGTCT
CGAGT (SEQ E) NO: 396)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODPAVSVALGOTVRVTCOGDGLRSYYASWYOOKPGOAPVLVMYGNNNRPSGIPDRF SGSSSGNT ASLTITGAOAEDEA VYYCNSRDSGANHLEVFGGGTKVTVL (SEQ ID NO: 397)
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGGTC
ACATGCCAAGGAGACGGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGG
GCAGGCCCCTGTCCTTGTCATGTATGGGAACAACAACCGGCCCTCAGGGATCCCAGACC
GATTCTCTGGCTCCAGCTCGGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGG
AAGATGAGGCTGTCTATTATTGTAATTCGCGGGACAGCGGTGCTAACCATCTGGAGGTTT
TCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 398)
Her2_SlR3Al_CS_9Cl
VH with CDR1 , CDR2 and CDR3 underlined
OVOLOESGPGLVKPSETLSLTCTVSGYSISSGYYWGWmOPPGRGLEWIGTIYHSGSTYYNPS LKSRLTISVDTSENOFSLKLSSVTAADTAVYYCARGIAGRTHYDYWGOGTMVTVSS (SEQ ID NO: 399)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCT
CACCTGCACTGTCTCTGGTTACTCCATTAGCAGTGGTTACTACTGGGGCTGGATCCGGCA
GCCCCCAGGGAGGGGGCTGGAGTGGATTGGGACTATCTATCATAGTGGGAGCACCTACT
ACAACCCGTCCCTCAAGAGTCGACTCACCATATCAGTAGACACGTCCGAGAACCAATTCT
CCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGAGGG
ATAGCAGGTCGGACCCATTATGACTACTGGGGCCAGGGGACAATGGTCACCGTCTCGAG
T (SEQ ID NO: 400)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPSSVSGAPGORVTISCSGSSSNIGAGYDVHWYOOLPGAAPKLLIYSNNHRPSGVPDR FSGSKSGTSASLAITGLOTEDEADYYCOSYDRSLSGRVFGGGTKLTVL (SEQ ID NO: 401)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTAACCAT
CTCCTGCAGTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGC
AGCTCCCAGGAGCAGCCCCCAAACTCCTCATCTATAGTAACAATCATCGGCCCTCAGGGG
TCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCT
CCAGACTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGAAGCCTGAGCGGTA
GGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 402) Her2_SlR2A_CS_5Al
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWYROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAOGTHSSGWSFGYWGOGTLVTVSS (SEQ ID NO: 403)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGCAGGG TACTCATAGCAGTGGCTGGTCCTTTGGGTACTGGGGCCAGGGCACCCTGGTCACCGTCTC GAGT (SEQ ID NO: 404)
VL with CDR1 , CDR2 and CDR3 underlined
LPVLTOPPSASGTPGORVTISCSGSSSNIGSKTVNWYOOLPGTTPKLLIYRNNORPSGVPDRFS GSKSGTSASLAISGLOSEDEADYYCAAWDDSLNGLIFGGGTKVTVL (SEQ ID NO: 405)
CTGCCTGTGCTGACTCAGCCCCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATC
TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAAAACTGTAAACTGGTACCAGCAGCTC
CCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAG
TCTGAGGATGAGGCTGATTATTATTGTGCAGCATGGGATGACAGCCTGAATGGTCTGATA
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 406)
Her2_SlR2A_CS_8C8
VH with CDR1 , CDR2 and CDR3 underlined
OVOLVOSGAE VKKPGESLKISCKTSGYSFTSYWIGWVROMPGKGLEWMGIIYPGDSDTRYSP SFOGOVTISADKSISTAYLOWSSLKASDTAMYYCARHDLPHOEYODNGMDVWGKGTLVTV SS (SEQ ID NO: 407)
CAGGTCCAGCTGGTACAGTCTGGAGCAGAGGTTAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGACTTCTGGATACAGCTTTACCAGCTATTGGATCGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGAT
ACAGCCCGTCTTTTCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAT
GACCTCCCCCATCAGGAGTATCAGGACAACGGTATGGACGTCTGGGGCAAAGGAACCCT
GGTCACCGTCTCGAGT (SEQ ID NO: 408)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVIOEPSFSVSPGETVTLTCALSSGSVSSSYWSWYOOTPGOAPRALIYNTNTRSSGVPDRF SGSILGNKAALTΓΓGAOADDESNYYCALYLGSGIWVFGGGTKLTVL (SEQ ID NO: 409)
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGAGACAGTCACACTC
ACTTGTGCCTTGAGCTCTGGCTCAGTCTCTAGTAGTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCGCGCTCATCTACAACACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTAACTATTACTGTGCGCTGTATCTGGGTAGTGGCATTTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 410) Her2_SlR3Al_CS_13H5
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKPGESLKISCKGSGYSFPSYWIGWVRQMPGKGLEWMGΠYPGDSETRYSP SFOGOVTISADKSISTAYLOWSSLKASDTAMYYCVRHLKPVAGPAWHDYGMDVWGOGTLV TVSS (SEQ ID NO: 411)
GAGGTCCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGATACAGCTTTCCCAGCTACTGGATCGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGAAACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGTGAGACAT
CTAAAACCAGTGGCTGGTCCCGCTTGGCACGACTACGGTATGGACGTCTGGGGCCAGGG
CACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 412)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVLOEPSISVSPGGWTLTCGLTSDSVSTTYYPSWYOOTPGOTPRTLSYSTNTRSSGVPDR FSGSILGNKAALTIAGAOADDEADYYCALYMGSGIWVFGGGTOLTVL (SEQ ID NO: 413)
CAGGCTGTGGTGCTCCAGGAGCCATCGATCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTAACCTCTGACTCAGTCTCGACTACTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGACTCCACGCACACTCAGCTACAGCACAAATACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCGCGGGGGCC
CAGGCAGATGATGAAGCTGATTATTACTGTGCCCTATATATGGGCAGTGGCATTTGGGTG
TTCGGCGGAGGGACCCAGCTCACCGTTTTA (SEQ ID NO: 414)
Her2_SlR2B_CS_5E9
VH with CDR1 , CDR2 and CDR3 underlined
OVOLVOSGAEVKKPGESLKISCKGSGYSFANYGIGWVROMPGKGLEWMGVIYPGDSDIRYS PSFOGOVIFSADRSISTAYLOWSSLKASDTAMYYCARHLSWLVGGNYGMDVWGKGTMVTV SS (SEQ ID NO: 415)
CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGATACAGTTTTGCCAACTACGGGATAGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGGTCATCTATCCTGGTGACTCTGATATCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCATCTTCTCAGCCGACAGGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTATTGTGCGAGACAT
CTCTCGTGGCTGGTCGGGGGGAACTACGGTATGGACGTCTGGGGCAAAGGGACAATGGT
CACCGTCTCGAGT (SEQ ID NO: 416)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVIOEPSFSVSPGGTVTLTCGLTSGSVSTSHYPSWYOOTPGOAPRTLIYSTNTRSSGVPGRF SGSILGNKAALTITGAOADDESDYYCVLYMGGGISVFGGGTKVTVL (SEQ ID NO: 417)
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGACCTCTGGCTCAGTCTCTACTAGTCACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGGTCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGACGATGAATCTGATTATTATTGTGTGCTGTATATGGGTGGTGGCATTTCGGTG
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 418) Her2_SlR3Al_CS_8F9
VH with CDR1, CDR2 and CDR3 underlined
OVOLVOSGAEVKKPGESLKISCKGSGYSFTSOWIAWVROMPGKGLEWMGΠYPGDSDTRYSP SFOGOVTISADKSINTAYLOWSSLKASDTAMYYCARHSGSSGDYYHYYGMDVWGOGTMVT VSS (SEQ ID NO: 419)
CAGGTGCAGCTGGTGCAATCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCCAGTGGATCGCCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACGAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAACACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAT
TCGGGGAGCTCTGGAGATTACTACCACTACTACGGTATGGACGTCTGGGGCCAAGGGAC
AATGGTCACCGTCTCGAGT (SEQ ID NO: 420)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVIOEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYOOTPGOAPRTLIYSTNTRSSGVPDRF SGSILGNKAALTITGAOADDESDYYCVLYMGSGISVFGGGTKLTVL (SEQ ID NO: 421 )
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTACTAGTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGCTGTATATGGGGAGTGGCATTTCGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 422)
Her2_SlR3Al_CS_14B5
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEMKKPGESLKISCKTSGYSFTGSWIAWVROMPGKGLEWMGIIYPGDSDTRYSP SFOGOVTISADKSISTAYLOWSSLKASDTAMYYCARIYSDSGYNWFDSWGRGTLVTVSS (SEQ ID NO: 423)
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAGATGAAAAAGCCCGGGGAGTCTCTGAAGAT
ATCCTGCAAGACTTCTGGATACAGCTTTACCGGCTCCTGGATCGCCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGACACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGATTT
ATAGTGACTCGGGTTACAATTGGTTCGACTCTTGGGGCAGGGGAACCCTGGTCACCGTCT
CGAGT (SEQ ID NO: 424)
VL with CDR1 , CDR2 and CDR3 underlined
OTWIOEPSFSVSPGGTVTLTCGLSSGSVSNSHYPSWYOOTPGO APRTLIYSTNTRSSGVPDRF SGSILGNKAALTITGAOADDESDFYCLLYLGSGISVFGGGTKLTVL (SEQ ID
NO: 425)
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTAATAGTCACTACCCCAGCTGGTATCAGCAG
ACCCCAGGCCAGGCTCCACGAACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCGGATGATGAATCTGATTTTTACTGTCTGCTATATCTGGGTAGTGGCATTTCGGTAT
TCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 426) Her2_SlR2A_CS_9E10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKPGESLKISCOGSGYTFASOWIAWVROMPGOGLEWMGTIWPGDSNPTYS PSFOGOVTISADKSISTAYLOWSSLKASDTAIYYCARLYNfNYPYFYGMDVWGOGTMVTVSS (SEQ ID NO: 427)
GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTCAGGGTTCTGGATACACCTTTGCCAGCCAATGGATCGCCTGGGTGCGCCAGAT
GCCCGGGCAAGGCCTGGAGTGGATGGGGACCATCTGGCCTGGTGACTCTAATCCCACAT
ATAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATCTATTACTGTGCGAGGCTCT
ACAATAACTATCCCTACTTCTACGGTATGGACGTCTGGGGCCAGGGGACAATGGTCACCG
TCTCGAGT (SEQ ID NO: 428)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVLOEPSFSVSPGGTVTLTCGLRSGSVSTTYYPSWYOOTPGOAPRTLIYSTNTRSSGVPDR FSGSIVGNKAALTITGAOADDESDYYCALYLGSGTWVFGGGTKLTVL (SEQ ID NO: 429)
CAGACTGTGGTGCTCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGGTCTGGCTCAGTCTCTACTACTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCGTCGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGCGCTATACCTGGGTAGTGGCACTTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 430)
Her2_SlR3Al_CS_7A10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKSGESLKISCKGSGYSFTSNWIGWVROMPGKGLEWMGΠYPGDSDTRYSP SFOGO VTISADKSVSTAYLOWSSLKASDTAMYYCARMLTDCSSTSCYSAGMDVWGKGTLV TVSS (SEQ ID NO: 431)
GAGGTCCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGTCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAATTGGATCGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCGTCAGCACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTATTGTGCGAGAATG
CTGACGGACTGTAGTAGTACCAGCTGCTATTCAGCCGGTATGGACGTCTGGGGCAAAGG
CACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 432)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVIOEPSFSVSPGGTVTLTCGLSSGSVSPSYYPSWYOOTPGOAPRTLIYSTNTRSSGVPDRF SGSILGNKAALTΓΓGAOADDESDYYCVLYMGSGSWVFGGGTKLTVL (SEQ ID NO: 433)
CAGGCTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGTTCTGGCTCAGTCTCTCCTAGTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACACTCATCTACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGCTGTATATGGGTAGTGGCTCTTGGGTG
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ BD NO: 434) Her2_SlR3Al_BMV_6H7
VH with CDR1 , CDR2 and CDR3 underlined
QVQLVESGGGWOPGRSLRLSCAASGFTFSSYAMNWVROAPGKGLEWVATISYDGSNKYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPAPYSSSGAFDIWGOGTLVTVSS (SEQ ID NO: 435)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGAACTGGGTCCGCCAGGC
TCCAGGCAAGGGGCTGGAGTGGGTGGCAACTATATCATATGATGGAAGCAATAAATACT
ACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACC
GGCCCCGTATAGCAGCTCCGGCGCTTTTGATATCTGGGGCCAAGGCACCCTGGTCACCGT
CTCTTCA (SEQ ID NO: 436)
VL with CDR1 , CDR2 and CDR3 underlined
DIOMTOSPSTLSASIGDRVTITCRASEGIYHWLAWYOOKPGKAPKLLTYKASSLASGAPSRFS GSGSGTDFTLTISSLOPDDFATYYCOOYSNYPLT FGGGTKLEΠCR (SEQ ID NO: 437)
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACC
ATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCA
GGGAAAGCCCCTAAACTCCTGACCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCA
AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGA
GGGACCAAGCTGGAGATCAAACGT (SEQ ID NO: 438)
Her2_SlR3Al_CS_12All
VH with CDR1 , CDR2 and CDR3 underlined
OVOLVOSGGEVKKPGESLKJSCKVSGDKFANYWIAWVROVPGRGLEWMGIIYPSDSDVRYS PSFOGOVTMSADKSTSTAYLOLSSLKASDTAMYYCAROVGGLVTTDTDSYFYGMDVWGOG TLVTVSS (SEQ ID NO: 439)
CAGGTCCAGCTGGTGCAGTCTGGAGGAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGTTTCTGGAGACAAGTTTGCCAACTACTGGATCGCCTGGGTGCGCCAGGT
GCCCGGGAGAGGCCTGGAGTGGATGGGAATCATCTATCCTAGTGACTCTGATGTCAGAT
ATAGTCCGTCCTTCCAAGGCCAAGTCACCATGTCAGCCGACAAGTCCACCAGCACCGCCT
ACTTGCAGTTGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAGACAG
GTGGGTGGACTGGTTACTACAGACACTGACTCCTACTTCTACGGCATGGACGTCTGGGGC
CAAGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 440)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVIOEPSFSVSPGGTVTLTCGLSSGPVSTSYYPSWFOOTPGO APRTLIYSTNTRSSGVPDRF SGSILGNKAALTrTGAOADDESDYYCVLYVGSGISLFGGGTKVTVL (SEQ ID NO: 441)
CAGGCTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCCCAGTCTCTACTAGTTACTACCCCAGCTGGTTCCAACAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGTTGTATGTGGGTAGTGGCATTTCGCTA
TTCGGCGGGGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 442) Her2_SlR3Al_CS_13D12
VH with CDR1 , CDR2 and CDR3 underlined
OVOLVOSGGEVKKPGESLKISCKVSGDSFTNYWIAWVROMPGRGLEWMGΠYPSDSDVRYS PSFOGOVTMSADKSISTAYLQLSSLKASDTAMYYCAROVGGLVTTDTDSYFYGMDVWGRG TLVTVSS (SEQ ID NO: 443)
CAGGTCCAGCTGGTGCAGTCTGGAGGAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGTTTCTGGAGACAGCTTTACCAACTACTGGATCGCCTGGGTGCGCCAGAT
GCCCGGGAGAGGCCTGGAGTGGATGGGAATCATCTATCCTAGTGACTCTGATGTCAGAT
ATAGTCCGTCCTTCCAAGGCCAGGTCACCATGTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTTGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAGACAG
GTGGGTGGACTGGTTACTACAGACACTGACTCCTACTTCTACGGCATGGACGTCTGGGGC
AGAGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 444)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVIOEPSFSVSPGGTVTLTCALNSGSVSTNYYPSWYOOTPGOAPRTLIHSTNTRSSGVPDRF SGSILGNNAALTITGAOAEDESDYYCALYMGSGISIFGGGTKLTVL (SEQ ID NO: 445)
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGCCTTGAACTCCGGCTCAGTCTCTACTAATTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCCACAGCACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAATGCTGCCCTCACCATCACGGGGGCC
CAGGCAGAGGATGAATCTGATTATTACTGTGCGCTATATATGGGTAGTGGCATTTCGATA
TTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 446)
Her2_SlR3Al_CS_7A8
VH with CDR1 , CDR2 and CDR3 underlined
OVOLOESGPGLVEPSETLSLTCSVSGGSISSSSSSWGWIROPPGKGLEWIGSIYYSGETYYNPS LKRRVTISTDTSKNOLSLELASVTAADTAVYYCAROVTSYGSDYFDYWGKGTLVTVSS (SEQ ID NO: 447)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGGAGCCTTCGGAGACCCTGTCCCT
CACCTGCAGTGTCTCTGGCGGCTCCATCAGCAGCAGTAGTTCCTCTTGGGGCTGGATCCG
CCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTACAGTGGAGAAACCT
ATTATAATCCGTCCCTCAAGAGGCGTGTCACCATATCCACAGACACGTCCAAGAACCAGT
TGTCCCTGGAGCTGGCCTCTGTGACCGCCGCAGACACGGCTGTATATTACTGTGCGAGGC
AAGTCACCAGTTATGGTTCTGACTACTTTGACTACTGGGGCAAAGGAACCCTGGTCACCG
TCTCGAGT (SEQ ID NO: 448)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVIOEPSFSVSPGGTVTLTCGLSSGSVSSNYYPSWYOOTPGOTPRTLIYNTNTRSSGVPDRF SGSILGNKAALTITGAOADDESDYYCVLYMGSGIRVFGGGTKVTVL (SEQ ID
NO: 449)
CAGGCTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTT
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTTCTAATTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGACTCCACGCACGCTCATCTACAACACAAACACTCGCTCTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGTTGTATATGGGTAGTGGCATTCGCGTG
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 450) Her2_SlR2A_CS_2C9
VH with CDR1, CDR2 and CDR3 underlined
EVQLVQSGAEVRKPGASVKVSCRSSGYTFTTYYLHWLROAPGQGLEWMGVINPSGGATAY AOSFOGRVTMTRDTATSTVYLDLSSLRTEDTAVYYCARSTPAEOLVPGFWGKGTMVTVSS (SEQ ID NO: 451)
GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGGCCTCAGTGAAGGT
TTCCTGCAGGTCATCTGGATACACCTTCACCACCTACTATTTGCACTGGCTACGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGAGTAATTAACCCTAGCGGCGGTGCCACAGCCT
ACGCGCAGAGTTTCCAGGGCAGAGTCACCATGACCAGGGACACGGCTACGAGCACAGTC
TATTTGGATCTGAGCAGCCTGAGAACTGAAGACACGGCCGTGTATTACTGTGCGAGATCC
ACCCCGGCGGAGCAGCTCGTCCCGGGCTTCTGGGGCAAAGGGACAATGGTCACCGTCTC
GAGT (SEQ ID NO: 452)
VL with CDR1 , CDR2 and CDR3 underlined
EIVMTOSPATLSVSPGDRATLSCRASOSVSTNVAWYOOKPGOPPRLLIYGASTRASGVPARFS GSGSGTEFTLTISSLOSEDFAVYYCOOYGDWPPITFGOGTRLEIKR (SEQ ID NO: 453)
GAAATTGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGACAGAGCCACC
CTCTCCTGCAGGGCCAGTCAGAGTGTTAGCACCAACGTAGCCTGGTACCAGCAGAAACC
TGGCCAACCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCTCTGGTGTCCCAGC
CAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGTCTGCAGTC
TGAAGATTTTGCAGTTTATTACTGTCAACAGTATGGTGACTGGCCTCCGATCACCTTCGG
CCAAGGGACACGACTGGAGATTAAACGT (SEQ ID NO: 454)
Her2_SlR3Al_CS_12Dl
VH with CDR1, CDR2 and CDR3 underlined
EVOLVESGGGLVOPGGSLKLSCAASGLNFDISTVHWVROASGKGLEWIGRIRSKAYNYATA YTESLKGRFIISRDESKNTADLOINSLKTEDTATYYCSMTFGDYYYYGMD VWGRGTLVTVSS (SEQ ID NO: 455)
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTAAAACT
TTCCTGTGCAGCCTCTGGGCTCAATTTCGATATCTCTACTGTGCACTGGGTCCGCCAGGCT
TCCGGGAAAGGGCTGGAGTGGATTGGCCGTATTAGAAGCAAAGCTTACAATTATGCGAC
AGCATATACTGAGTCGCTGAAGGGCAGGTTCATCATCTCCAGAGATGAGTCAAAGAATA
CGGCGGATCTGCAAATCAACAGCCTGAAAACCGAGGACACGGCCACATATTACTGTAGT
ATGACCTTCGGTGACTACTACTACTACGGCATGGACGTCTGGGGCCGGGGCACCCTGGTC
ACCGTCTCGAGT (SEQ ID NO: 456)
VL with CDR1 , CDR2 and CDR3 underlined
OA VLTOPSSVSGAPGORVTITCTGSSSNIGAGYD VHWYOOLPGTAPKLLIYSNSYRPSGVSDR FSGSKSGTSASLVIAGLOAEDEADYYCOSYDSSHWFFGGGTKLTVL (SEQ ID NO: 457)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
ACCTGCACTGGAAGCAGCTCCAACATCGGGGCCGGTTACGATGTTCACTGGTACCAGCA
ACTTCCAGGAACAGCCCCCAAACTCCTCATCTATAGTAATTCTTATCGGCCCTCTGGGGT
CTCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCGCTGGACTC
CAGGCTGAGGATGAGGCTGATTATTACTGTCAGTCCTATGACAGCAGTCATTGGTTTTTC
GGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 458) Her2_SlR2A_CS_7D4
VH with CDR1 , CDR2 and CDR3 underlined
QVQLQQSGAEVKKPGASVKVSCKVSGYTPPDLSIHWVROAPGEGLEWMGRFDFEDGETINA OKFOGRVTMTADTSTNTGYMEVSSLRFEDTAVYYCATTLRFSGYYYGMDFWGRGTLVTVS S (SEQ ID NO: 459)
CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT
CTCCTGCAAGGTTTCCGGATACACCCCCCCTGATTTATCCATCCACTGGGTGCGACAGGC
TCCTGGAGAAGGGCTTGAGTGGATGGGACGTTTTGATTTTGAAGATGGTGAAACAATCA
ACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCGCGGACACATCCACAAACACAGGC
TACATGGAGGTGAGCAGCCTGAGATTTGAGGACACGGCCGTGTATTACTGTGCAACAAC
ACTTCGATTTTCTGGTTACTACTACGGTATGGACTTCTGGGGCCGAGGAACCCTGGTCAC
CGTCTCGAGT (SEQ ID NO: 460)
VL with CDR1 , CDR2 and CDR3 underlined
OAVVIOEPSLSVSPGGTVTLTCALSSGSVSTGYYPSWYOOTPGO APRTLIYNTDTRSSGVPGR FSGSILGDKAALTITGAOADDESDYYCVLYMGSGIWVFGGGTKVTVL (SEQ ID NO: 461 )
CAGGCTGTGGTGATCCAGGAGCCATCGCTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGCCTTGAGCTCTGGCTCAGTCTCCACTGGTTACTACCCCAGCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATTTACAACACAGACACTCGCTCTTCTGGGGTC
CCTGGTCGCTTCTCTGGCTCCATCCTTGGGGACAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGCTGTATATGGGTAGTGGCATTTGGGTG
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 462)
Her2_SlR3Al_CS_15B8
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKPGESLKISCKGSGYNFNTHWIGWVROMPGKGLEWMGLIYPDDSDTRFS PSFEGOVTLSADRSISTAYLOWTSLKASDTAMYYCARYKKSSGYYTGYGMDVWGRGTMVT VSS (SEQ ID NO: 463)
GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGATACAACTTTAACACTCACTGGATCGGGTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGCTCATCTACCCTGATGACTCTGACACCCGAT
TCAGCCCGTCCTTCGAAGGCCAGGTCACCCTCTCAGCCGACAGGTCCATCAGTACCGCCT
ACCTGCAGTGGACCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGATAC
AAAAAAAGTAGTGGTTATTACACAGGATATGGTATGGACGTCTGGGGCCGAGGGACAAT
GGTCACCGTCTCGAGT (SEQ ID NO: 464)
VL with CDR1 , CDR2 and CDR3 underlined
OTVVIQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPTWYOOTPGOAPRTLIYSTNSRFSGVPDRF SGSILGSKAALTITGAOADDESDYYCVLYMGSGISVFGGGTKVTVL (SEQ ID NO: 465)
CAGACTGTGGTGATCCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTC
ACTTGTGGCTTGAGCTCTGGCTCAGTCTCTACTAGTTACTACCCCACCTGGTACCAGCAG
ACCCCAGGCCAGGCTCCACGCACGCTCATCTATAGCACAAACAGTCGCTTTTCTGGGGTC
CCTGATCGCTTCTCTGGCTCCATCCTTGGGAGCAAAGCTGCCCTCACCATCACGGGGGCC
CAGGCAGATGATGAATCTGATTATTACTGTGTGCTATATATGGGTAGTGGCATTTCGGTG
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ED NO: 466) Her2_S6R3_DP47_lA10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCATSLGYGDFDYWGRGTTVTVSS (SEQ ED NO: 467)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAACTTC GTTGGGTTACGGTGACTTTGACTACTGGGGGCGAGGGACCACGGTTACCGTCTCGAGT (SEQ ID NO: 468)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPSSASGAPGHRVIISCSGSSSNIGSYYVSWYOOLPGAAPKLLIYRNDERPSGVP ARFS GSTSGTSASLAISGLHSEDEADYYCAAWDDSLNGPVFGGGTKVTVL (SEQ ID NO: 469)
CAGGCTGTGCTGACTCAGCCGTCCTCAGCGTCTGGGGCCCCCGGGCACAGGGTCATCATC
TCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTTATTATGTAAGCTGGTACCAGCAGCTC
CCAGGAGCGGCCCCCAAACTCCTCATCTATCGTAATGATGAGCGGCCCTCAGGGGTCCCT
GCCCGATTCTCTGGCTCCACGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAC
TCTGAGGATGAGGCTGATTATTATTGTGCAGCATGGGATGACAGCCTGAATGGTCCGGTT
TTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 470)
Her2_S6R2_DP47_lEl 1
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAVHGYGDSVDDALDVWGRGTLVTVS S (SEQ ID NO: 471)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGGTCCA
TGGCTACGGAGACTCCGTGGATGATGCTCTTGATGTCTGGGGCCGAGGAACCCTGGTCAC
CGTCTCGAGT (SEQ ID NO: 472)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSASGTPGOTISISCSGSNSNIGTYSVSWYOOLPRAAPRLLWANDRRPSGVPDRFS GSKSGTSASLAISGLOSEDEADYYCAVWDDRLNGFVFGTGTKLTVL (SEQ ED NO: 473)
CAGTCTGTGCTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGACGATCTCCATC
TCTTGTTCTGGAAGCAACTCCAACATCGGAACTTATAGTGTTAGCTGGTACCAGCAGCTC
CCACGAGCGGCCCCCAGACTCCTCGTCTATGCTAATGATCGCCGGCCCTCAGGGGTCCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAG
TCTGAGGATGAGGCTGATTATTACTGTGCAGTATGGGATGACAGGTTGAATGGTTTTGTC
TTCGGAACTGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 474) Her2 S5R2_DP47_1H11
VH with CDR1, CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDDDFWSGYPFLYYYYGMDVWGRG TMVTVSS (SEQ ID NO: 475)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTG-GTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGA
TGACGATTTTTGGAGTGGTTATCCATTCCTCTACTACTACTACGGTATGGACGTCTGGGGC
CGAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 476)
VL with CDR1 , CDR2 and CDR3 underlined
OSVVTOPPSASGTPGORVTISCSGTSSNIGSNAVNWYOOLPGTAPKLLIYNNNORPSGVPDRF SGSKSGTSASLAISGLOSEDEADYYCAAWDDSLNVYWFGGGTKLTVL (SEQ ID
NO: 477)
CAGTCTGTCGTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCAT
CTCTTGTTCTGGAACTAGTTCCAACATCGGAAGTAATGCTGTAAACTGGTACCAGCAACT
CCCAGGAACGGCCCCCAAACTCCTCATCTATAATAATAATCAGCGGCCCTCAGGGGTCCC
TGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGACTCCA
GTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGTTTATGT
GGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 478)
Her2_S6R3_CS_lG5
VH with CDR1 , CDR2 and CDR3 underlined
OVOLOESGPGLVKPSETLSLTCTVSGGSΓΓSDLSYWGWLROPPGKGLEWIASGGDGESTYYN PSLNGRVTFSVDTPKNOFSLRLSSVTAADTAVYYCARHPLYYCSGGRCYSGNFDFWGOGTL VTVSS (SEQ ID NO: 479)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCT
CACCTGCACTGTCTCTGGTGGCTCCATCACCAGTGATCTTTCCTACTGGGGCTGGCTCCGC
CAGCCCCCCGGGAAGGGTCTGGAGTGGATTGCGAGTGGTGGTGACGGTGAGAGCACCTA
CTACAACCCGTCCCTCAACGGTCGAGTCACCTTTTCCGTGGACACGCCCAAGAACCAATT
CTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTATATTACTGTGCGAGACA
CCCACTCTACTATTGTAGTGGTGGTCGCTGCTACTCCGGGAACTTTGACTTTTGGGGCCA
GGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 480)
VL with CDR1 , CDR2 and CDR3 underlined
OA VLTOPSSASGTPGORVTISCSGTTPNIGSNFVYWYOOLPGTAPKLLIYRNEORPSGVPVRFS GSKSGTSASLAISDLRSEDEADYYCAAWDDSLSGVVFGGGTKLTVL (SEQ ID NO: 481)
CAGGCTGTGCTGACTCAGCCGTCCTCAGCGTCTGGGACCCCCGGTCAGAGGGTCACCATT
TCTTGTTCTGGAACGACCCCCAATATTGGAAGTAATTTTGTCTACTGGTATCAACAACTCC
CAGGGACGGCCCCCAAACTCCTCATCTACAGGAATGAGCAGCGCCCTTCAGGGGTCCCT
GTCCGATTCTCTGGCTCCAAGTCTGGCACATCAGCCTCCCTGGCCATCAGTGACCTCCGG
TCCGAGGATGAGGCTGACTATTACTGTGCAGCGTGGGATGACAGCCTGAGTGGTGTGGT
CTTCGGCGGGGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 482) Her2_S6R2_DP47_lHl 1
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKYGGYDADAFDVWGRGTMVTVSS (SEQ ID NO: 483)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAGTA
TGGTGGCTACGACGCTGATGCCTTTGATGTCTGGGGCCGAGGGACAATGGTCACCGTCTC
GAGT (SEQ ID NO: 484)
VL with CDR1 , CDR2 and CDR3 underlined
OSVVTOPPSVSAAPGOKVTISCSGSSSNIGDYYVSWYOOLPGTAPTLLIYDNDKRPSEVPDRF SGSKSGTSATLGITGLOTGDEADYYCTSWDSSLSAGVFGGGTKVTVL (SEQ ID NO: 485)
CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATC
TCCTGCTCTGGAAGTAGCTCCAACATTGGAGATTATTATGTATCCTGGTACCAGCAACTC
CCAGGAACGGCCCCCACACTCCTCATTTATGACAATGATAAGCGACCCTCAGAAGTTCCT
GACCGATTCTCTGGCTCCAAGTCTGGCACGTCGGCCACCCTCGGCATCACCGGACTCCAG
ACTGGGGACGAGGCCGATTATTACTGCACTTCATGGGATAGCAGCCTGAGTGCTGGGGT
GTTCGGCGGAGGGACCAAGGTCACCGTCCTA (SEQ ID NO: 486)
Her2_S5R3_DP47_lA10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKWGWDYYDTTGHDAFDFWGRGTM VTVSS (SEQ ID NO: 487)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACTTACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCTAAATG
GGGCTGGGATTACTATGACACAACTGGTCATGATGCCTTTGATTTCTGGGGCCGGGGGAC
AATGGTCACCGTCTCGAGT (SEQ ID NO: 488)
VL with CDR1 , CDR2 and CDR3 underlined
OAVLTOPSSVSGAPGORVTISCTGSSSNIGAD YYVNWYOOLPGKAPEIVIFNDDNRPSGVPNR FSGSKSGTSASLAITGLOAEDEADYYCOSYDSVLSAYVFGTGTKVTVL (SEQ ID NO: 489)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGATTATTATGTAAATTGGTATCAGCAA
CTTCCAGGAAAAGCCCCCGAAATCGTAATTTTTAATGATGACAATCGGCCCTCAGGGGTC
CCTAACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTC
CAGGCTGAAGATGAGGCTGATTATTATTGCCAGTCTTATGACAGTGTCCTGAGTGCTTAT
GTCTTCGGAACTGGGACCAAGGTCACCGTCCTA (SEQ DD NO: 490) Her2_S5R2_DP47_lDl 1
VH with CDR1 , CDR2 and CDR3 underlined
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSAISGSGGSTYYAD SVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARNSSNWSGAFDIWGRGTTVTVSS (SEQ ID NO: 491)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATACCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAAGGAA
TAGCAGCAATTGGAGTGGTGCTTTTGATATCTGGGGGCGGGGGACCACGGTCACCGTCTC
GAGT (SEQ ID NO: 492)
VL with CDR1 , CDR2 and CDR3 underlined osvLTOPPsvsGAPGORVTisciGTHSNiGAGY A VNWΎOOLPGTAPKLLIYGNNNRPSGVPDR
FSGSKSGTSASLAINGLOADDESDYYCOSYDASLRVLFGGGTKLTVL (SEQ ID NO: 493)
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCAT
CTCCTGCATTGGAACCCACTCAAACATCGGGGCAGGTTACGCTGTGAACTGGTACCAGCA
GCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAATAACAATCGGCCCTCAGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAATGGGCT
CCAGGCTGACGATGAGTCTGATTATTATTGCCAGTCCTATGACGCCAGTCTGAGAGTTTT
ATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 494)
Her2_S5R2_CS_lA8
VN with CDR1 , CDR2 and CDR3 underlined
EVOLVOSGAEVKKPGESLKISCKGSGYSFSNYWIGWVROMPGKGLEWMGΠYPGDSDTRYSP SFOGOVTISADKSISTAYLOWSSLKASDSAMYYCARHDSTMGYDAFHMWGOGTLVTVSS
(SEQ ID NO: 495)
GAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGAT
CTCCTGTAAGGGTTCTGGGTACAGCTTTAGTAACTACTGGATCGGCTGGGTGCGCCAGAT
GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGAT
ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGTACCGCCT
ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACAGTGCCATGTATTACTGTGCGAGACAT
GATTCGACTATGGGATATGATGCTTTTCATATGTGGGGCCAAGGAACCCTGGTCACCGTC
TCGAGT (SEQ ED NO: 496)
VL with CDR1 , CDR2 and CDR3 underlined
OA VLTOPSSVSGAPGORVTISCTGSSSNIGAGYD VHWYOOLPGTAPKLLIYGNSNRPSGVPDR FSGSKSGTSASLATTGLOAEDEADYYCOSYDSSLSGPVVFGGGTKLTVL (SEQ ED NO: 497)
CAGGCTGTGCTGACTCAGCCGTCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCA
GCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCT
CCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGCCC
TGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 498) Her2_S6R3_CS_lB7
VH with CDR1 , CDR2 and CDR3 underlined
QVOLVOSGAEIKKPGESLKISCEGSGYRFΓSHWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP SFOGOVTISADKSISTAYLOWSSLKASDTAMYYCARHSATHDAFDIWGRGTLVTVSS
(SEQ ID NO: 499)
CAGGTGCAGCTGGTGCAGTCTGGGGCAGAAATAAAAAAGCCGGGGGAGTCTCTGAAGAT CTCCTGTGAGGGTTCTGGATACAGGTTTACCAGCCACTGGATCGGCTGGGTGCGCCAGAT GCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGAT ACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAT AGTGCGACGCATGATGCTTTTGATATCTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 500)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPPSVSGAPGORVTISCSGSSSNIGTGYD VHWYOOLPGTAPKLLIYSFNKRPSGVPDRF SASKSGTSASLVITGLOAEDEAD YYCOSYDNLSGPHVVFGTGTKLTVL (SEQ ID
NO: 501)
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC
TCCTGTAGTGGGAGCAGCTCCAACATCGGGACAGGTTACGATGTTCACTGGTACCAGCA
ACTTCCAGGAACAGCCCCCAAACTCCTCATCTATAGTTTCAATAAGCGGCCCTCAGGGGT
CCCTGACCGGTTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCACTGGGCTC
CAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAATTTGAGTGGTCCCCAT
GTGGTTTTCGGCACAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 502)
Her2_S6R2_CS_lE5
VH with CDR1 , CDR2 and CDR3 underlined
EVOLVETGAEVKKPGASMKVSCKASGYSFTDYYMHWVROAPGOGLEWMGWINPNSGDTN YAOKFOGRVTMTRDTSITTAYMELSRLRSDDTAVYYCATERYNSGWEWGRGTLVTVSS (SEQ ID NO: 503)
GAGGTGCAGCTGGTGGAGACTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAATGAAGGT CTCCTGCAAGGCTTCTGGATACAGCTTCACCGACTACTATATGCACTGGGTGCGACAGGC CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAATAGTGGTGACACAAACT ATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCACCACAGCC TACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTATATTACTGTGCGACAGA GAGGTATAACAGTGGCTGGGAATGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 504)
VL with CDR1 , CDR2 and CDR3 underlined
OSALTOPASVSGSPGOSmSCTGTSSDVGAYNYVSWYOOHPGKAPKLMIYDVTTRPSGVSNR FSGSKSGNTASLTISGLOAEDEADYYCTSYTRSSTWFGGGTKLTVL (SEQ ID NO: 505)
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCT
CCTGCACTGGAACCAGCAGTGACGTTGGTGCTTATAACTATGTCTCCTGGTACCAACAAC
ACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCACTACTCGGCCCTCAGGGGTTT
CTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCC
AGGCTGAGGACGAGGCTGATTATTACTGCACCTCATATACTCGCAGCAGCACTGTGGTCT
TCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 506) Her2_S6R3_BMV_lC2
VH with CDR1, CDR2 and CDR3 underlined
KVOLVOSGSELKKPGASVKVSCOASGYTITNHSMNWVROAPGOGLEWMGWINTNTGNPTY AQGFTGRFVFSLDTSANTATLQΓΓNVQAEDTAVYYCAREGSIDVSGTP YYYGMDAWGOGTT VTVSS (SEQ ID NO: 507)
AAGGTGCAGCTGGTGCAGTCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGT
TTCCTGCCAGGCTTCTGGATACACCATCACTAACCATAGCATGAATTGGGTGCGACAGGC
CCCTGGGCAAGGGCTTGAGTGGATGGGATGGATCAACACCAACACTGGGAACCCTACGT
ATGCCCAGGGCTTCACAGGACGGTTTGTCTTCTCCTTGGACACCTCTGCCAACACGGCAA
CTTTGCAGATCACCAACGTGCAGGCTGAGGACACAGCCGTCTACTACTGTGCGAGAGAG
GGGAGTATAGACGTGTCTGGAACGCCCTACTACTACGGAATGGACGCCTGGGGGCAAGG
GACCACGGTCACCGTCTCCTCA (SEQ ID NO: 508)
VL with CDR1 , CDR2 and CDR3 underlined
OSVLTOPASVSGSPGOSITISCTGTSSDVGGYNYVSWYOOHPGKAPKLMIYEGSERPSGVPNR FSGSKSGNTASLTISGLOAEDEADYYCSSYTTRSTRVFGGGTKLTIL (SEQ ID NO: 509)
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCT
CCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAAC
ACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTGAGCGGCCCTCAGGGGTT
CCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTC
CAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAACCAGGAGCACTCGAGT
TTTCGGCGGAGGGACCAAGCTGACCATCCTA (SEQ ID NO: 510)
Her2_S5R2_DP47_lB10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARVGAGENYYHYYIMDVWGRGTLVT VSS (SEQ ID NO: 511)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGI GGGGGCCGGGGAGAACTACTACCACTACTACATCATGGACGTCTGGGGCCGGGGCACCC TGGTCACCGTCTCGAGT (SEQ ID NO: 512)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODP AVSVALGORVRVTCOGDSLRGYYASWYOOKPGO APVLVIYGENNRPSGIPDRFS GSSSGNTASLTIIGAOAEDEADYYCNSRHSSGNYLVFGGGTKLTVL (SEQ ID NO: 513)
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGAGAGTCAGGGTC ACATGCCAAGGAGACAGCCTCAGAGGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGG ACAGGCCCCTGTTCTTGTCATTTATGGTGAAAACAACCGGCCCTCAGGGATCCCAGACCG ATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCATTGGGGCTCAGGCGGA AGATGAGGCTGACTATTACTGTAACTCCCGGCACAGCAGTGGTAATTATCTGGTGTTCGG CGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 514) Her2_S6R3_DP47_lC12
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARGFGDYWGRGTMVTVSS (SEQ ID NO: 515)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC
TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT
ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGG
CTTTGGTGACTACTGGGGCCGGGGGACAATGGTCACCGTCTCGAGT (SEQ ID
NO: 516)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODPAVSVALGOTVRΓΓCOGDSLRSYYASWYOOKPGOAPVLVIYAKNNRPSGIPDRFS GSDSGNTASLTITGAOAEDEADYYCLSRDSSGNHLVFGGGTKLTVL (SEQ ID NO: 517)
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATC
ACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGG
ACAGGCCCCTGTACTTGTCATCTATGCTAAAAACAACCGACCCTCAGGGATCCCAGACCG
ATTCTCTGGCTCCGACTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGA
AGATGAGGCTGACTATTACTGTCTCTCCCGGGACAGCAGTGGTAACCATCTGGTATTCGG
CGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 518)
Her2_S5R2_DP47_lD10
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARTTADAFDIWGRGTMVTVSS (SEQ ID NO: 519)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAAC TACGGCAGATGCTTTTGATATCTGGGGCAGAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO: 520)
VL with CDR1 , CDR2 and CDR3 underlined
SSELTODPAVSVALGOTVKITCOGDSLRNYYAGWYOOKPGOAPVLVIYGENKRPSGIPDRFS GSNSGNTASLTLTGAOAEDEADYYCNSRDSSSNLVVFGGGTKLTVL (SEQ ID NO: 521 )
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAAGATC
ACATGCCAAGGCGACAGCCTCAGAAACTATTATGCAGGCTGGTACCAGCAGAAGCCAGG
ACAGGCCCCTGTACTTGTCATCTATGGTGAAAACAAGCGGCCCTCAGGGATCCCTGACCG
ATTCTCTGGCTCCAACTCAGGAAACACAGCTTCCTTGACCCTCACTGGGGCTCAGGCGGA
AGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTAGTAACCTCGTGGTATTCGG
CGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 522) Her2_S6R3_DP47_lH9
VH with CDR1 , CDR2 and CDR3 underlined
EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTYYA DSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARTTADAFDIWGRGTTVTVSS (SEQ ID NO: 523)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACT ACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAAC TACGGCAGATGCTTTTGATATCTGGGGGAGGGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO: 524)
VL with CDR1 , CDR2 and CDR3 underlined ssELTODP AvsvALGOTvsiTCOGDSLRNFYASWYLOKPGO APILVΓYGKNKRPSGΓPDRVSG
SSSEDTASLTITGAOAEDEADYYCNSRDSSGNWFGGGTKLTVL (SEQ ID NO: 525)
CTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCCTGGGACAGACAGTCAGCATCA
CATGCCAGGGAGACAGCCTCAGAAACTTTTATGCAAGCTGGTACCTGCAGAAGCCAGGA
CAGGCCCCAATACTTGTCATCTATGGTAAAAACAAGCGGCCCTCTGGGATCCCAGACCG
AGTCTCTGGCTCCAGCTCAGAAGACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGA
AGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACGTGGTCTTCGGCG
GGGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 526)
>HerSMIP_leader_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggt (SEQ ID NO: 527)
>HerSMIP_Protein_leader
MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 528)
>HerSMIP_G4Sx3_linker_CDS ggaggcggcggttcaggcggaggtggctctggcggtggcggaagt (SEQ ID NO: 529)
>HerSMIP_Protein_G4Sx3_linker
GGGGSGGGGSGGGGS (SEQ ID NO: 530)
>HerSMIP_SCCP_Hinge_CDS gagcccaaatcttctgacaaaactcacacatgcccaccgtgccca (SEQ ID NO: 531)
>HerSMIP_Proteiii_SCCP_Hinge
EPKSSDKTHTCPPCP (SEQ ID NO: 532)
>HerlOl_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaagtgcagctggtgcagtctggggctgaggtgagg aagcctggggcctcagtgaaggtttcctgcaggtcatctggatacaccttcaccacctactatttgcactggctacgacaggcccctggacaagggc ttgagtggatgggagtaattaaccctagcggcggtgccacagcctacgcgcagagtttccagggcagagtcaccatgaccagggacacggctac gagcacagtctatttggatctgagcagcctgagaactgaagacacggccgtgtattactgtgcgagatccaccccggcggagcagctcgtcccgg gcttctggggcaaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacttgaaat tgtgatgacgcagtctccagccaccctgtctgtgtctccaggggacagagccaccctctcctgcagggccagtcagagtgttagcaccaacgtagc ctggtaccagcagaaacctggccaacctcccaggctcctcatctatggtgcatccaccagggcctctggtgtcccagccaggttcagtggcagtgg gtctgggacagagttcactctcaccatcagcagtctgcagtctgaagattttgcagtttattactgtcaacagtatggtgactggcctccgatcaccttc ggccaagggacacgactggagattaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaact cctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt gagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgacc aagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaact acaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgt cttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 533)
>Herl01_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVRKPGASVKVSCRSSGYTFTTYYLHWLRQAP GQGLEWMGVINPSGGATAYAQSFQGRVTMTRDTATSTVYLDLSSLRTEDTAVYYCARSTPA EQLVPGFWGKGTMVTVSSGGGGSGGGGSGGGGSALEIVMTQSP ATLSVSPGDRATLSCRAS QSVSTNV AWYQQKPGQPPRLLIYGASTRASGVP ARFSGSGSGTEFTLTISSLQSEDF AVYYCQ QYGDWPPITFGQGTRLEDCRD VREPKS SDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK. (SEQ ID NO: 534)
>HerlO2_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcagatgcagctggtgcagtctggggctgaggtgaag aagccaggggcctcagtgaaagtctcctgcaagtcttctggatacaccttcaaggactactatatcaactgggtgcgacaggcccctggacaaggg cttgagtgggtgggatggatcaaccctaaaaatggtatcacaaaatattcgcagaattttcagggcagggtctccatgaccacggatacgtccatca gcacagtctacatggacctgagaggtctgacatctgacgacacggccgtttattattgtgcgagagacgcgaaccgccttagggtgggctggttcg acccctggggccaaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggct gtgctgactcagccgtcctcagtgtctgggtccccagggcagagggtcagcatctcctgcactgggagcagctccaacatcggggcaggttatgat gtacattggtatcaacaacttccaggaacagcccccaaactcctcatctacggtaacatcaatcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcactgggctccaggctgaagatgaggctgattattactgccagtcctatgacagcagcctgagggct gcggtattcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag cacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac aaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggat gagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 535)
>Herl02_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQMQLVQSGAEVKKPGASVKVSCKSSGYTFKDYYINWVRQA PGQGLEWVGWINPKNGITKYSQNFQGRVSMTTDTSISTVYMDLRGLTSDDTA VYYCARD AN RLRVGWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSGSPGQRVSISCTGSS SNIGAGYD VHWYQQLPGTAPKLLIYGNINRPSGVPDRFSGSKSGTSASLAITGLQAEDEAD YY CQSYDSSLRAAVFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGP S VFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVK-FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKE YKCKVSNKALP APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK. (SEQ ID NO: 536) >HerlO3_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgcagggtactcatagcagtggctggtcctttgggt actggggccagggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactgcctgtgc tgactcagcccccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagtaaaactgtaaact ggtaccagcagctcccaggaacgacccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccgattctctggctccaagt ctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattattgtgcagcatgggatgacagcctgaatggtctgata ttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctga actcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 537)
>Herl03_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAQGTHS
SGWSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSALPVLTQPPSASGTPGQRVTISCSGSSSNI
GSKTVNWYQQLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAA
WDDSLNGLEFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN
GKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNΉYTQ
KSLSLSPGK. (SEQ ED NO: 538)
>HerlO4_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtacagctgcagcagtcaggggctgaggtgaag aagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccccccctgatttatccatccactgggtgcgacaggctcctggagaaggg cttgagtggatgggacgttttgattttgaagatggtgaaacaatcaacgcacagaagttccagggcagagtcaccatgaccgcggacacatccaca aacacaggctacatggaggtgagcagcctgagatttgaggacacggccgtgtattactgtgcaacaacacttcgattttctggttactactacggtat ggacttctggggccgaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagg ctgtggtgatccaggagccatcgctctcagtgtcccctggagggacagtcacactcacttgtgccttgagctctggctcagtctccactggttactacc ccagctggtaccagcagaccccaggccaggctccacgcacgctcatttacaacacagacactcgctcttctggggtccctggtcgcttctctggctc catccttggggacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgctgtatatgggtagtggcatttggg tgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 539)
>Herl04_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGQVQLQQSGAEVKKPGASVKVSCKVSGYTPPDLSIHWVRQAP GEGLEWMGRFDFEDGETENAQKFQGRVTMTADTSTNTGYMEVSSLRFEDTAVYYCATTLRF SGYYYGMDFWGRGTLVTVSSGGGGSGGGGSGGGGSAQAWIQEPSLSVSPGGTVTLTCALS SGSVSTGYYPSWYQQTPGQAPRTLEYNTDTRSSGVPGRFSGSELGDKAALTITGAQADDESDY YCVLYMGSGIWVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 540)
>HerlO5_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtccagctggtacagtctggagcagaggttaaaa agcccggggagtctctgaagatctcctgtaagacttctggatacagctttaccagctattggatcggctgggtgcgccagatgcccgggaaaggcc tggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtcttttcaaggccaggtcaccatctcagccgacaagtccatcagc accgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagacatgacctcccccatcaggagtatcaggacaa cggtatggacgtctggggcaaaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgc acagactgtggtgatccaggagccatcgttctcagtgtcccctggagagacagtcacactcacttgtgccttgagctctggctcagtctctagtagtta ctaccccagctggtaccagcagaccccaggccaggctccacgcgcgctcatctacaacacaaacactcgctcttctggggtccctgatcgcttctct ggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctaactattactgtgcgctgtatctgggtagtggca tttgggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccca gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaa caaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggga tgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 541)
>Herl05_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGAEVKKPGESLKISCKTSGYSFTSYWIGWVRQMP GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHDLPH QEYQDNGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSAQTVVIQEPSFSVSPGETVTLTCAL SSGSVSSSYYPSWYQQTPGQAPRALIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESN YYCALYLGSGIWVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 542)
>HerlO6_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggagcagaggtgaag aagcccggggagtctctgaagatctcctgtcagggttctggatacacctttgccagccaatggatcgcctgggtgcgccagatgcccgggcaagg cctggagtggatggggaccatctggcctggtgactctaatcccacatatagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatc agcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatctattactgtgcgaggctctacaataactatccctacttctacggtat ggacgtctggggccaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaga ctgtggtgctccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgaggtctggctcagtctctactacttactacc ccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacagcacaaacactcgctcttctggggtccctgatcgcttctctggct ccatcgtcgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgcgctatacctgggtagtggcacttg ggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca cctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 543) >Herl06_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCQGSGYTFASQWIAWVRQMP GQGLEWMGTIWPGDSNPTYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAIYYCARLYNNY PYFYGMD VWGQGTMVTVSSGGGGSGGGGSGGGGSAQTVVLQEPSFSVSPGGTVTLTCGLR SGSVSTTYYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSIVGNKAALTΓΓGAQADDESDY YCALYLGSGTWVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVK-FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNWSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 544)
>HerlO7_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcagatgcagctggtgcagtctggggctgaggtgaag aagcctggggcctcagtgaaggtttcctgcaaggcatctggatacaccttcaccagctactatatacactgggtgcgacaggcccctggacagggc cctgagtggatgggaataatcctccctagtggtggcagcaccagctacgcacaggagttccagggcagactctccatgaccagggacacgtccac gagcacagtgtacatggagctgagcgacctgagatctgacgacacggccatttattattgtgcgagagactatgataggagtgcttatcttgatatctg gggccgagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgttgac gcagccgccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacatcggaagaaatactgtaaactggt acaagcagttcccaggaacggcccccaaactcctcatctatagtgataataagcggccctcagggatccctgaccgattctctggctccaagtctgg cacctcagcctccctggccatcagtgggctccagtctggggatgaggctgattattactgtgccgcatgggatgacagcctgaatggccatgtggta ttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctga actcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 545)
>Herl07_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAP
GQGPEWMGIILPSGGSTSYAQEFQGRLSMTRDTSTSTVYMELSDLRSDDTAIYYCARDYDRS
AYLDIWGRGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTPGQRVTISCSGSSSNIG
RNTVNWYKQFPGTAPKLLIYSDNKRPSGIPDRFSGSKSGTSASLAISGLQSGDEADYYCAAW
DDSLNGHVVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK. (SEQ ID NO: 546)
>HerlO8_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaagtgcagctggtgcagtctggggctgaggtgaag aagcctggggcctcagtgagggtctcctgcaagggttctggaaacaccttcaccggccactacatccactgggtgcgacaggcccctggacaag gacttgagtggctgggatggatcgaccctaacactggtgacatacagtattcagaaaactttaagggctcggtcaccttgaccagggacccatccat caactcagtcttcatggacctgatcaggctgacatctgacgacacggccatgtattactgtgcgagagaaggtgccgggctcgccaactactattact acggtctggacgtctggggccgagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagt gcacagactgtggtgctccaggagccttcgttctcagtgtcccctggggggacagtcacactcacttgtggcttgaactttggctcagtctctactgct tactaccccagttggtaccagcagaccccaggccaagctccacgcacgctcatctacggcacaaatattcgttcctctggggtcccggatcgcttct ctggctccatcgtagggaacaaagctgccctcaccatcacgggggcccagacagaagatgagtctgattattattgtgcgctgtatatgggtagtgg catgctcttcggcggcgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag cacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac aaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggat gagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 547)
>Her 108_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGASVRVSCKGSGNTFTGHYIHWVRQAP
GQGLEWLGWIDPNTGDIQYSENFKGSVTLTRDPSINSVFMDLIRLTSDDTAMYYCAREGAGL
ANYYYYGLDVWGRGTMVTVSSGGGGSGGGGSGGGGSAQTVVLQEPSFSVSPGGTVTLTCG
LNFGSVSTAYYPSWYQQTPGQAPRTLIYGTNIRSSGVPDRFSGSIVGNKAALTITGAQTEDES
DYYCALYMGSGMLFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKI7NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ
DWLNGKΈYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 548)
>HerlO9_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgccagagatttaactggcccaaaccactggttcttcga tctctgggggcgggggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgt gctgactcagccaccctccgtgtccgtgtccccaggacagacagccagcatctcctgctctggacatagattgggcgataagtatgttagttggtatc ggcagaggccgggccagtcccctgtgctggtcatctatcaagatgagaagaggtactcagggatctctgagcgattctctggctccaactctggga acgtagccactctgaccatcaccgggacccaggctatggatgaggctgactatcactgtcaggcgtgggacagcaccactgtggtgttcggcgga gggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggg tggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcac gtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccc ccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaacc aggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 549)
>Herl09_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLTG PNHWFFDLWGRGTTVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSVSPGQTASISCSGHRL GDKYVSWYRQRPGQSPVLVIYQDEKRYSGISERFSGSNSGNV ATLTITGTQAMDEADYHCQ AWDSTTVVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKJTSΓWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK. (SEQ ID NO: 550)
>HerllO_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagaggcagtagagtggggacgatttggggaag ccttgacgactggggcaaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcact tgaaacgacactcacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtagtagcagca gctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatgctgcatccagcagggccactggcgtcccagacaggttca gtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcacgg ttcaccttcggccaagggacacgactggagattaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagc acctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg gtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagga gcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaaca aagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatg agctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccgg agaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 551)
>Her 110_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSICNTLYLQMNSLRAEDTAVYYCARGSRV
GTIWGSLDDWGKGTMVTVSSGGGGSGGGGSGGGGSALETTLTQSPGTLSLSPGERATLSCR
ASQSSSSSYLAWYQQKPGQAPRLLIYAASSRATGVPDRFSGSGSGTDFTLTISRLEPEDFAVY
YCQQYGSSRFTFGQGTRLEIKRD VREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK. (SEQ JD NO: 552)
>Herlll_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtgcagtctggagcagaggtgaaa aagcccggggagtctctgaagatctcctgtaagggttctggatacagttttgccaactacgggataggctgggtgcgccagatgcccgggaaagg cctggagtggatgggggtcatctatcctggtgactctgatatcagatacagcccgtccttccaaggccaggtcatcttctcagccgacaggtccatca gcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattattgtgcgagacatctctcgtggctggtcggggggaactac ggtatggacgtctggggcaaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgca cagactgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgacctctggctcagtctctactagtcac taccccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacagcacaaacactcgctcttctggggtccctggtcgcttctct ggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagacgatgaatctgattattattgtgtgctgtatatgggtggtggcat ttcggtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag cacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac aaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggat gagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 553)
>Herlll_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGQVQLVQSGAEVKKPGESLKISCKGSGYSFANYGIGWVRQMP
GKGLEWMGVIYPGDSDIRYSPSFQGQVIFSADRSISTAYLQWSSLKASDTAM YYCARHLSWL
VGGNYGMDVWGKGTMVTVSSGGGGSGGGGSGGGGSAQTVVIQEPSFSVSPGGTVTLTCGL
TSGSVSTSHYPSWYQQTPGQAPRTLIYSTNTRSSGVPGRFSGSILGNKAALTΓΓGAQADDESD
YYCVLYMGGGISVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKJTVFWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 554) >Herll2_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggcagaggtgaaa cagcccggggagtctctgaagatctcctgtaagggttctggatacagctttagcaactactggatcggctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctgatgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaggtccatc agcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccacgtattactgtgcgagaggaaatgttataaatggaaataccaatgct tttgatatctgggggcgggggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacag gctgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctactggttactac cccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacaacacaaacagtcgctcttctggggtccctgatcgcttctctggc tccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgctgtatatgggtagtggcatttcg gtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacc tgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 555)
>Herl 12_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKQPGESLKISCKGSGYSFSNYWIGWVRQMP
GKGLEWMGΠYPDDSDTRYSPSFQGQVTISADRSISTAYLQWSSLKASDTATYYCARGNVING
NTNAFDIWGRGTTVTVSSGGGGSGGGGSGGGGSAQAWIQEPSFSVSPGGTVTLTCGLSSGS
VSTGYYPSWYQQTPGQAPRTLIYNTNSRSSGVPDRFSGSILGNKAALTITGAQADDESDYYC
VLYMGSGISVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVUNAKTKPREEQYNSTYRWSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK. (SEQ ID NO: 556)
>Herll3_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgctgctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctatcagtggtagtggtggtagcacatactacgcagactccgcgaagggccggttcaccatctccagagacaattccaag aacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagggacagggtctctaactggaactactacgg ccaggacagctactttgactactggggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggc ggaagtgcacaggctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaacat cggaagtaattatgtatactggtaccagcaactcccaggaacggcccccaaagtcctcatctataggaataatcagcggccctcaggggtccctga ccgattctctggctccaagtctggcacctcagcgtccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcatcatgggatg gcagcctgagtggtccggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgc ccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagt gcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc ccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagag caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga (SEQ ID NO: 557)
>Herl 13_Protein_Ieader-stop
MEAP AQLLFLLLLWLPDTTGEVLLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVSAISGSGGSTYYADSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRVSN WNYYGQDSYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSAQAVLTQPPSASGTPGQRVTIS CSGSSSNIGSNYVYWYQQLPGTAPKVLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDE AD YYCASWDGSLSGPVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPK DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL HQDWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ ID NO: 558)
>Herll4_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctgggggaggcttggta cagcctgggggatccctgagactctcctgtgcagcctctggattcacctttagcagctatcccatgcactgggtccgccaggctccaggcaagggg ctggagtgggtggcagttgtatcgttcgatggatctaagaaatactctgcagactccgtgaagggccgattcaccatctccagagacatctccaaga acacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtatattactgtgcgaaagatcgctatgattcggggactttctactacggc atggacgtctggggccggggcaccctggtcaccgtctcgagtggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgcagtct gccctgactcagcctgcctccgtgtctggatctcgtggacagtcgatcaccatctcctgcactggaaccactggtgacgttggtggttatgactatgtc tcctggtaccaacagcacccaggcagagcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgatcgcttctctgcctcca agtccggcaatacggcctccctgaccatctctggactccaggctgaggatgaggctgattatttctgcagcacatatgcaccccccggtattattatgt tcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctga actcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 559)
>Her 114_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYPMHWVRQAP GKGLEWV AWSFDGSKKYSADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAKDRYD SGTFYYGMD VWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSRGQSITISCTGTT GDVGGYDYVSWYQQHPGRAPKLLIYGNSNRPSGVPDRFSASKSGNTASLTISGLQAEDEADY FCSTYAPPGIIMFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKJNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKE YKCKVSNKALP APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYT QKSLSLSPGK. (SEQ ID NO: 560)
>Herll5_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagtctgggggaggcttggta cagcctggggggtccctgagactctcctgtgctgcctctggattcacctttgacagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcaattattagtggtagagatggttacacatactacacagactccgtgaagggtcggttcaccatctccagagacaattccaagaa cacggtgtatctgcaaatgaacagcctgagagccgaggacacgggtgtgtattattgtgcgagaaatggggagtggcccggaatcttagactactg gggcagggggacaatggtcaccgtctcctcaggtggaggcggttcaggcggaggtggcagcggcggtggcggatcggacatccagatgaccc agtctccttccaccctgtctgcatctattggagacagagtcaccatcacctgccgggccagtgagggtatttatcactggttggcctggtatcagcaga agccagggaaagcccctaaactcctgatctataaggcctctagtttagccagtggggccccatcaaggttcagcggcagtggatctgggacagatt tcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacaatatagtaattatccgctcactttcggcggagggaccaagct ggagatcaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcag tcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctg aggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaa aaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcct gacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcc cgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgat gcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 561) >Herl 15_Protein_Ieader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLRLSCAASGFTFDSYAMSWVRQAP GKGLEWVSΠSGRDGYTYYTDSVKGRFTISRDNSKNTVYLQMNSLRAEDTGVYYCARNGEW PGILDYWGRGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTΓΓCRASEGIYH WLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSN
YPLTFGGGTKLEIKRD VREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVK-FhTVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALP APΠEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK. (SEQ ID NO: 562)
>Herll6_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtggagtctgggggaggcgtggtc cagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatgctatgaactgggtccgccaggctccaggcaagggg ctggagtgggtggcaactatatcatatgatggaagcaataaatactacgcagactccgtgaagggccgattcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagaccggccccgtatagcagctccggcgcttttg atatctggggccaaggcaccctggtcaccgtctcttcaggtggaggcggttcaggcggaggtggcagcggcggtggcggatcggacatccagat gacccagtctccttccaccctgtctgcatctattggagacagagtcaccatcacctgccgggccagtgagggtatttatcactggttggcctggtatca gcagaagccagggaaagcccctaaactcctgacctataaggcctctagtttagccagtggggccccatcaaggttcagcggcagtggatctggga cagatttcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacaatatagtaattatccgctcactttcggcggagggac caagctggagatcaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggac cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaag accctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtacc gtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatc gagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc agcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacg cctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctcc gtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 563)
>Herl 16_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVESGGGWQPGRSLRLSCAASGFTFSSYAMNWVRQAP GKGLEWV ATISYDGSNKYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARP APY SSSGAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTΓΓCRASEGIY HWLAWYQQKPGKAPKLLTYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDF ATYYCQQYS NYPLTFGGGTKLEDCRD VREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK. (SEQ ID NO: 564)
>Herll7_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcagatgcagctggtgcagtctggggcagaggtgaaa aagcccggggagtctctgaagatgtcctgtaagggttctggatacagctttaccagctactggatcggctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctggtaactccgataccagatacaacccgtccttcgaaggccaggtcaccatctcagccgacaagtccatc aacaccgccttcctgcagtggaacagcctgaaggcctcggacaccgccatatattattgtgcgcgggctccctgggtgggtgcttttgatacttggg gccaggggacaatggtcaccgtctcttcaggtggaggcggttcaggcggaggtggcagcggcggtggcggatcggacatcgtgatgacccagt ctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagggtattagtagctggttggcctggtatcagcagaaa ccagggagagcccctaaggtcttgatctataaggcatctactttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttca ctctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtaccccgtggacgttcggccaagggaccaagctg gagatcaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcagt cttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctga ggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggt cagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctg acctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatg catgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 565)
>Herl 17_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQMQLVQSGAEVKKPGESLKMSCKGSGYSFTSYWIGWVRQM PGKGLEWMGΠYPGNSDTRYNPSFEGQ VTISADKSINTAFLQWNSLKASDTAIYYCARAP WV GAFDTWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSTLSASVGDRVTΓΓCRASQGISS WLAWYQQKPGRAPKVLIYKASTLESGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQSYST PWTFGQGTKLEIKRD VREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK. (SEQ ID NO: 566)
>Herll8_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtcgggcccaggactggtg gagccttcggagaccctgtccctcacctgcagtgtctctggcggctccatcagcagcagtagttcctcttggggctggatccgccagcccccaggg aaggggctggagtggattgggagtatctattacagtggagaaacctattataatccgtccctcaagaggcgtgtcaccatatccacagacacgtcca agaaccagttgtccctggagctggcctctgtgaccgccgcagacacggctgtatattactgtgcgaggcaagtcaccagttatggttctgactacttt gactactggggcaaaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggct gtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacacttacttgtggcttgagctctggctcagtctcttctaattactacccca gctggtaccagcagaccccaggccagactccacgcacgctcatctacaacacaaacactcgctcttctggggtccctgatcgcttctctggctccat ccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgttgtatatgggtagtggcattcgcgtgtt cggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaa ctcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 567)
>Herl 18_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGQVQLQESGPGLVEPSETLSLTCSVSGGSISSSSSSWGWIRQPPG KGLEWIGSIYYSGETYYNPSLKRRVTISTDTSKNQLSLELASVTAADTAVYYCARQVTSYGSD YFD YWGKGTLVTVSSGGGGSGGGGSGGGGSAQAVVIQEPSFSVSPGGTVTLTCGLSSGSVSS NYYPSWYQQTPGQTPRTLIYNTNTRSSGVPDRFSGSILGNKAALTΓΓGAQADDESDYYCVLY MGSGIRVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK. (SEQ ID NO: 568)
>Herll9_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtccagctggtgcagtctggggcagaggtgaaa aagtccggggagtctctgaagatctcctgtaagggttctggatacagctttaccagtaattggatcggctgggtgcgccagatgcccgggaaaggc ctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccgtca gcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattattgtgcgagaatgctgacggactgtagtagtaccagctgct attcagccggtatggacgtctggggcaaaggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcg gaagtgcacaggctgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagttctggctcagtctct cctagttactaccccagctggtaccagcagaccccaggccaggctccacgcacactcatctacagcacaaacactcgctcttctggggtccctgat cgcttctctggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgctgtatatgggt agtggctcttgggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccacc gtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac atgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccg cgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaag gtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgg gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 569)
>Herl 19_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKSGESLKISCKGSGYSFTSNWIGWVRQMP GKGLEWMGIIYPGDSDTRYSPSFQGQ VTISADKSVSTAYLQWSSLKASDTAMYYCARMLTD CSSTSCYSAGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSAQA WIQEPSFSVSPGGTVTLT CGLSSGSVSPSYYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDE SDYYCVL YMGSGSWVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ ID NO: 570)
>Herl20_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggcagaggtgaaa aagcccggggagtctctgaagatctcctgtaagggctctggatacagctttaccagctattggatcggctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatc agcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgttttactgtgcgagactcaatgatagtagtggttatacgactaact ttgactactggggccaaggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagg ctgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctactcgttacaacc ccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacagtacaaacactcgttcttctggggtccctgaccgcttctctggctc catccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgcgctgtatatgggtagtggcatttggg tgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 571)
>Herl20_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMP GKGLEWMGΠYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMFYCARLNDSS GYTTNFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAQAWIQEPSFSVSPGGTVTLTCGLSSG SVSTRYNPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYC
ALYMGSGIWVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKI^NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 572) >Herl21_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtgcaatctggggcagaggtgaaa aagcccggggagtctctgaagatctcctgtaagggttctggatacagctttaccagccagtggatcgcctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctggtgactctgatacgagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatc aacaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagacattcggggagctctggagattactacca ctactacggtatggacgtctggggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcgg aagtgcacagactgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctcta ctagttactaccccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacagcacaaacactcgctcttctggggtccctgatc gcttctctggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatggatctgattattactgtgtgctgtatatgggg agtggcatttcggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccacc gtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac atgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccg cgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaag gtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgg gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 573)
>Her 12 l_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGAEVKKPGESLKISCKGSGYSFTSQWIAWVRQMP GKGLEWMGΠYPGDSDTRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAMYYCARHSGSS GDYYHYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSAQTVVIQEPSFSVSPGGTVTLTC GLSSGSVSTSYYPSWYQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDG SDYYCVLYMGSGISVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGP S VFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALH NHYTQKSLSLSPGK. (SEQ ID NO: 574)
>Herl22 CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtcgggcccaggactggtg aagccttcggagaccctgtccctcacctgcactgtctctggttactccattagcagtggttactactggggctggatccggcagcccccagggaggg ggctggagtggattgggactatctatcatagtgggagcacctactacaacccgtccctcaagagtcgactcaccatatcagtagacacgtccgaga accaattctccctgaagctgagttctgtgaccgccgcagacacggccgtgtattactgtgcgagagggatagcaggtcggacccattatgactactg gggccaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtgctga ctcagccgtcctcagtgtctggggccccagggcagagggtaaccatctcctgcagtgggagcagctccaacatcggggcaggttatgatgtacac tggtaccagcagctcccaggagcagcccccaaactcctcatctatagtaacaatcatcggccctcaggggtccctgaccgattctctggctccaagt ctggcacctcagcctccctggccatcactgggctccagactgaggatgaggctgattattactgccagtcctatgacagaagcctgagcggtaggg tgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 575)
>Herl22_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGQVQLQESGPGLVKPSETLSLTCTVSGYSISSGYYWGWIRQPPG RGLEWIGTIYHSGSTYYNPSLKSRLTISVDTSENQFSLKLSSVTAADTAVYYCARGIAGRTHY DYWGQGTMVTVSSGGGGSGGGGSGGGGSAQA VLTQPSSVSGAPGQRVTISCSGSSSNIGAG YDVHWYQQLPGAAPKLLIYSNNHRPSGVPDRFSGSKSGTSASLAΓΓGLQTEDEADYYCQSYD RSLSGRVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK. (SEQ ED NO: 576)
>Herl23_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtccagctggtgcagtctggggctgaggtgaag aagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccagctatggtatcagctgggtgcgacaggcccctggacaaggg cttgagtggatgggatggatcagcgcttacaatggtaacacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccac gagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagtggggtcgggatattgtagtggtggta gctgctacgtgggctggttcgacccctggggccgggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcg gtggcggaagtgcactttcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaagatcacatgccaaggagacagcct cagtgcctattatgcaacctggtaccagcagaagccaggccaggcccctgtacttgtcatctatggtaaaaacaagcggccgtccgggatcccaga ccgattctctggctccaagtcaggaaacacagcttccttgaccatcacgggggctcaggcggaagatgaggctgactattactgtaactcccggga cagcagtggtaatgatcattatgtcttcggaactgggaccaagctgaccgttctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacat gcccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagac aaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaa gtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccct gcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggag agcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaa gagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgg gtaaatga (SEQ ID NO: 577)
>Herl23_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAP GQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVG SGYCSGGSCYVGWFDPWGRGTMVTVSSGGGGSGGGGSGGGGSALSSELTQDP AVSV ALGQ TVKΓΓCQGDSLSAYYATWΎQQKPGQ AP VLVIYGKNKRPSGIPDRFSGSKSGNTASLTITGAQA EDEAD YYCNSRDSSGNDHYVFGTGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKJ=T^WYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALP APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK. (SEQ ID NO: 578)
>Herl24_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggcagaagtcaag aggcccggagagtctctgaagatctcctgtagggcctctggatacatctttacgaacaattgggtcgcctgggtgcgccagcagcccgggaaagg cctggagtggatggggatcatctatcctggtgactctgacaccagatacagcccgtccttccaaggccaggtcactttctcggccgacacgtccatc aacaccgcctacctacagtggaatagcctgaaggcctcggacaccgccacttacttctgtgcgcgagaggcctacaactcatacgaatattacggt atggacgtctgggggcgagggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcaca gactgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctactaattacta ccccagctggtaccagcagaccccaggccaggctccacgcacgctcatctacaacacaaacactcgctcttctggggtccctgatcgcttctctgg ctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgctgtatatgggtagtggcatttc ggtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca cctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ EO
NO: 579) >Herl24_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKRPGESLKISCRASGYIFRNNΛVVAWVRQQP GKGLEWMGΠYPGDSDTRYSPSFQGQVTFSADTSINTAYLQWNSLKASDTATYFCAREAYNS YEYYGMDVWGRGTTVTVSSGGGGSGGGGSGGGGSAQTWIQEPSFSVSPGGTVTLTCGLSS GSVST^[YYPSWYQQTPGQAPRTLIY^RMTRSSGVPDRFSGSILG^IKAALTITGAQADDESDYY
CVLYMGSGISVFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALP APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 580)
>Herl25_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtccagctggtgcagtctggaggagaggtgaaa aagcccggggagtctctgaagatctcctgtaaggtttctggagacaagtttgccaactactggatcgcctgggtgcgccaggtgcccgggagagg cctggagtggatgggaatcatctatcctagtgactctgatgtcagatatagtccgtccttccaaggccaagtcaccatgtcagccgacaagtccacca gcaccgcctacttgcagttgagcagcctgaaggcctcggacaccgccatgtattactgtgcaagacaggtgggtggactggttactacagacactg actcctacttctacggcatggacgtctggggccaaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggt ggcggaagtgcacaggctgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggccca gtctctactagttactaccccagctggttccaacagaccccaggccaggctccacgcacgctcatctacagcacaaacactcgctcttctggggtcc ctgatcgcttctctggctccatccttgggaacaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgttgtatgt gggtagtggcatttcgctattcggcggggggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccc accgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaag ccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagca atgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa tga (SEQ ID NO: 581)
>Herl25_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGQVQLVQSGGEVKKPGESLKISCKVSGDKFANYWIAWVRQVP GRGLEWMGΠYPSDSDVRYSPSFQGQVTMSADKSTSTAYLQLSSLKASDTAMYYCARQVGG LVTTDTDSYFYGMDVWGQGTLVTVSSGGGGSGGGGSGGGGSAQAWIQEPSFSVSPGGTVT LTCGLSSGPVSTSYYPSWFQQTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQAD
DESDYYCVLYVGSGISLFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKP KDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK. (SEQ ID NO: 582)
>Herl26_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagtccgggggaggcttggtc cagccgggggggtccctaaaactttcctgtgcagcctctgggctcaatttcgatatctctactgtgcactgggtccgccaggcttccgggaaagggc tggagtggattggccgtattagaagcaaagcttacaattatgcgacagcatatactgagtcgctgaagggcaggttcatcatctccagagatgagtca aagaatacggcggatctgcaaatcaacagcctgaaaaccgaggacacggccacatattactgtagtatgaccttcggtgactactactactacggc atggacgtctggggccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcaca ggctgtgctgactcagccgtcctcagtgtctggggccccagggcagagggtcaccatcacctgcactggaagcagctccaacatcggggccggt tacgatgttcactggtaccagcaacttccaggaacagcccccaaactcctcatctatagtaattcttatcggccctctggggtctctgaccgattctctg gctccaagtctggcacctcagcctccctggtcatcgctggactccaggctgaggatgaggctgattattactgtcagtcctatgacagcagtcattgg tttttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacct gaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 583)
>Herl26_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLVQPGGSLKLSCAASGLNFDISTVHWVRQAS
GKGLEWiGRiRSKA YNYATAYTESLKGRFΠSRDESKNTADLQINSLKTEDTATYYCSMTFGD
YYYYGMD VWGRGTLVTVSSGGGGSGGGGSGGGGSAQA VLTQPSSVSGAPGQRVTITCTGSS SNIGAGYDVHWYQQLPGTAPKLL]YSNSYRPSGVSDRFSGSKSGTSASLVIAGLQAEDEADY YCQSYDSSHWFFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKΓ^NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 584)
>Herl27_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtccagctggtgcagtctggaggagaggtgaaa aagcccggggagtctctgaagatctcctgtaaggtttctggagacagctttaccaactactggatcgcctgggtgcgccagatgcccgggagaggc ctggagtggatgggaatcatctatcctagtgactctgatgtcagatatagtccgtccttccaaggccaggtcaccatgtcagccgacaagtccatcag caccgcctacctgcagttgagcagcctgaaggcctcggacaccgccatgtattactgtgcaagacaggtgggtggactggttactacagacactga ctcctacttctacggcatggacgtctggggcagaggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggt ggcggaagtgcacagactgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtgccttgaactccggctca gtctctactaattactaccccagctggtaccagcagaccccaggccaggctccacgcacgctcatccacagcacaaacactcgctcttctggggtc cctgatcgcttctctggctccatccttgggaacaatgctgccctcaccatcacgggggcccaggcagaggatgaatctgattattactgtgcgctatat atgggtagtggcatttcgatattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgc ccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagt gcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc ccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagag caatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga (SEQ ID NO: 585)
>Her 127_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGQVQLVQSGGEVKKPGESLKISCKVSGDSFTNYWIAWVRQMP GRGLEWMGIIYPSDSDVRYSPSFQGQ VTMSADKSISTAYLQLSSLKASDTAMYYCARQVGGL VTTDTDSYFYGMDVWGRGTLVTVSSGGGGSGGGGSGGGGSAQTWIQEPSFSVSPGGTVTL TCALNSGSVSTNYYPSWYQQTPGQAPRTLIHSTNTRSSGVPDRFSGSILGNNAALTITGAQAE DESDYYCAL YMGSGISIFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKJT^WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPFFIKTISK^AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ ID NO: 586)
>Herl28_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtccagctggtgcagtctggagcagaggtgaaa aagcccggggagtctctgaagatctcctgtaagggttctggatacagctttcccagctactggatcggctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctggtgactctgaaaccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatc agcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgtgagacatctaaaaccagtggctggtcccgcttgg cacgactacggtatggacgtctggggccagggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggc ggaagtgcacaggctgtggtgctccaggagccatcgatctcagtgtcccctggagggacagtcacactcacttgtggcttaacctctgactcagtct cgactacttactaccccagctggtaccagcagaccccaggccagactccacgcacactcagctacagcacaaatactcgctcttctggggtccctg atcgcttctctggctccatccttgggaacaaagctgccctcaccatcgcgggggcccaggcagatgatgaagctgattattactgtgccctatatatg ggcagtggcatttgggtgttcggcggagggacccagctcaccgttttaagtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccca ccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtc acatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagc cgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgca aggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaa tgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagca ggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaat ga (SEQ ID NO: 587)
>Herl28_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCKGSGYSFPSYWIGWVRQMP GKGLEWMGΠYPGDSETRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRHLKPV AGPAWHDYGMDVWGQGTLVTVSSGGGGSGGGGSGGGGSAQAVVLQEPSISVSPGGTVTLT CGLTSDSVSTTYYPSWYQQTPGQTPRTLSYSTNTRSSGVPDRFSGSILGNKAALTIAGAQADD EAD YYCALYMGSGIWVFGGGTQLTVLSDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKJTS[WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ DD NO: 588)
>Herl29_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtccagctggtacagtctggggctgaggtgagg aagcctggggcctcagtcaaggtctcctgcagggcttctggatacaacttcaaagactactatttgcactgggtgcgccaggcccctggagaaggg cttgagtggatggggtggatcaaccctcacgctggtaccacaaaatatgcacagaattttcagcacaggattattatgaccagggacacgaccatca ccacagcctacatggaactgagcagtctgaaatctgacgacacagccatttatttctgtaccagatactactttgacagtagtggttattttaggttcgac ccctggggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtc gtgacgcagccgccctcagtgtctggggccccaggacagaaggtcaccatctcctgctctggaagcagctccaacattgggaataattatgtatcct ggtaccagcagctcccaggaacagcccccaaactcctcatttatgacaataataagcgaccctcagggattcctgaccgattctctggctccaagtct ggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattattactgcggaacatgggatagcagcctgagtgctggg gtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacc tgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 589)
>Herl29_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVRKPGASVKVSCRASGYNFKDYYLHWVRQA
PGEGLEWMGWINPHAGTTKYAQNFQHRΠMTRDTTITTAYMELSSLKSDDTAIYFCTRYYFD
SSGYFRFDPWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVVTQPPSVSGAPGQKVTISCSGSS
SMG>JNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGΓΓGLQTGDEADYYC
GTWDSSLSAGVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 590) >Herl30_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggcagagatgaaa aagcccggggagtctctgaagatatcctgcaagacttctggatacagctttaccggctcctggatcgcctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctggtgactctgacaccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatc agcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgaggatttatagtgactcgggttacaattggttcg actcttggggcaggggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagactg tggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctaatagtcactacccca gctggtatcagcagaccccaggccaggctccacgaacgctcatctacagcacaaacactcgctcttctggggtccctgatcgcttctctggctccat ccttgggaacaaagctgccctcaccatcacgggggcccaggcggatgatgaatctgatttttactgtctgctatatctgggtagtggcatttcggtatt cggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaa ctcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 591)
>Herl30_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEMKKPGESLKISCKTSGYSFTGSWIAWVRQMP GKGLEWMGΠYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDTAMYYCARJYSDSG YNWFDSWGRGTLVTVSSGGGGSGGGGSGGGGSAQTWIQEPSFSVSPGGTVTLTCGLSSGSV SNSHYPSWYQQTPGQ APRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDFYCLL YLGSGISVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKJTSTVVRRVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK. (SEQ ID NO: 592)
>Herl31_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaagtgcagctggtgcagtctggagcagaggtgaaa aagcccggggagtctctgaagatctcctgtaagggttctggatacaactttaacactcactggatcgggtgggtgcgccagatgcccgggaaaggc ctggagtggatggggctcatctaccctgatgactctgacacccgattcagcccgtccttcgaaggccaggtcaccctctcagccgacaggtccatc agtaccgcctacctgcagtggaccagcctgaaggcctcggacaccgccatgtattactgtgcgagatacaaaaaaagtagtggttattacacagga tatggtatggacgtctggggccgagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagt gcacagactgtggtgatccaggagccatcgttctcagtgtcccctggagggacagtcacactcacttgtggcttgagctctggctcagtctctactag ttactaccccacctggtaccagcagaccccaggccaggctccacgcacgctcatctatagcacaaacagtcgcttttctggggtccctgatcgcttct ctggctccatccttgggagcaaagctgccctcaccatcacgggggcccaggcagatgatgaatctgattattactgtgtgctatatatgggtagtggc atttcggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccca gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaa caaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggga tgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 593)
>Herl31_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCKGSGYNFNTHWIGWVRQMP GKGLEWMGLIYPDDSDTRFSPSFEGQVTLSADRSISTAYLQWTSLKASDTAM YYCARYKKSS GYYTGYGMDVWGRGTMVTVSSGGGGSGGGGSGGGGSAQTWIQEPSFSVSPGGTVTLTCG LSSGSVSTSYYPTWYQQTPGQAPRTLIYSTNSRFSGVPDRFSGSILGSKAALTITGAQADDESD YYCVLYMGSGISVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKI7NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 594)
>Herl32_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtccggcccaggactcgtg aagcctgcggggactctgtccctcacctgcgctgtctccggtgactccatcagcagcaatcactggtggaattgggtccgccagtccccagggaag ggactggaatggattggtgaaatctttcatagtgacattcgcatcctcaacccgtccctcaagaggcgcgtctccatgtcagtcgacaggtccaagg accaattctccctgcaactgacctctgtgaccgccgcggacacggccgtgtattactgtgcgagaggtttccatggtgactccggcagaggacttga cacctggggcagaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttcttct gagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcagggtcacatgccaaggagacggcctcagaagttattatgcaagctgg taccagcagaagccagggcaggcccctgtccttgtcatgtatgggaacaacaaccggccctcagggatcccagaccgattctctggctccagctc gggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgtctattattgtaattcgcgggacagcggtgctaaccatctgga ggttttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac ctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggt ggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 595)
>Herl32_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLQESGPGLVOAGTLSLTCAVSGDSISSNHWWNWVRQS PGKGLEWIGEIFHSDIRILNPSLKRRVSMSVDRSKDQFSLQLTSVTAADTA VYYCARGFHGDS GRGLDTWGRGTLVTVSSGGGGSGGGGSGGGGSALSSELTQDPAVSVALGQTVRVTCQGDG LRSYYASWYQQKPGQAPVLVMYGNNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEAVYYC NSRDSGANHLEVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 596)
>Herl33_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagtctgggggaggcttggca cagcctggggggtccctgagactctcctgtgcagcctctggattaacctttaccacctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcaagtattagtggaagtggtcatagcacatattacgcagactccgtgaagggccgcttcaccatctccagagacatttccaaga acacgttgtatctgcaaatgaacagcctcagagccgaggacacggccgtctattactgtgcgaaagattcgtcggcttttgggtttgtacacggtgctt ttgatatctggggccagggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttct tctgagctgactcaggaccctgctgcgtctgtggccttgggacagacagtcagcatcacatgccaaggagacagcctcagaaactattgggctagc tggtaccagcagaagccaggacaggcccctgtacttgtcatctatggtaaaaatacccggccctcagggatcccagaccgattctctggctccacct caggaaacacagcttccttgaccatcactggggctcaggcggaggatgaggctgactattactgcaactcccgggacagtggtcaccgtcttctttt cggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaa ctcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 597) >Herl33_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVESGGGLAQPGGSLRLSCAASGLTFTTYAMSWVRQAP GKGLEWVSSISGSGHSTYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAKDSSAF
GFVHGAFDiwGQGTLVTvssGGGGSGGGGSGGGGSALSSELTQDP AASVALGQTVSΓΓCQG
DSLRNYWASWYQQKPGQAPVLVIYGKNTRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYY CNSRDSGHRLLFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFlNrVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 598)
>Herl34_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagtccggagcagaagtcaaa aagcccggagagtctctgaagatctcctgtaaggcttctggatacatctttacgaacaattggatcgcctgggtgcggcagcagcccgggaaaggc ctggagtggatgggaatcatctatcctggtgactctgacaccagatacagcccgtccttccagggccgggtcactttctcagccgacacgtccatca acaccgcctacctccagtggagtagcctgaaggcctcggacaccgccacttactactgtgcgagagaggcctacaactcatacgagtactacggt atggacgtctggggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcaca gactgtggtgctccaggagccagcgttctcagtgtcccctggagggacagtcacactcacctgtggcttgagctctggctcagtctctactagttact accccagttggtaccagcagaccccaggccagcctccacgcacgctcatctacaacacaaacacccgctcttctggggtctctgatcgcttctctgg ctccatccttgggaacaaagctgccctcaccatcacgggggcccaggccgaagatgaatctgattattactgtgttctgtatatgggtagtggcatttc ggtattcggcggggggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca cctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 599)
>Herl34_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLVESGAEVKKPGESLKISCKASGYIFTNNWIAWVRQQPG KGLEWMGΠYPGDSDTRYSPSFQGRVTFSADTSINTAYLQWSSLKASDTATYYCAREAYNSY EYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSAQTWLQEPAFSVSPGGTVTLTCGLSS GSVSTSYYPSWYQQTPGQPPRTLIYNTNTRSSGVSDRFSGSILGNKAALTΓΓGAQAEDESDYY CVLYMGSGISVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 600)
>Herl35_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaaagatttctggtttggactaccaccttccttctttg actcttggggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgt gctgactcagccaccctcagtgtccgtgtccccaggacagaaggccagcatcacctgctctggagaaagaatgggggataaatatgctgcctggt atcagcagaagccaggccagtcacctatactggtcatctatcaagatacaaagcggccctcagggatccctgagcgattctctggctccaactctgg gaacacagccacgttgaccatcagcgggacccaggacatggatgaggctgactattactgtcaggtgtgggacagcagcactggggtattcggc ggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcct gggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgag ccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacag cacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct catgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 601)
>Herl35_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDFWF
GLPPSFFDSWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSVSPGQKASITCSGER
MGDKYAAWYQQKPGQSPELVIYQDTKRPSGIPERFSGSNSGNTATLTISGTQDMDEADYYCQ
VWDSSTGVFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
K^YKCKVSNK-ALPAPmKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNfYKTTPPVLDSDGSFFLYSKXTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK. (SEQ ID NO: 602)
>Herl36_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacatggccgtgtattactgtgcgaggactcccgggtatagcagtggctggtactcgg tttggggccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtcgt gacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgtactgggagcagctccaacatcggggcagggtatgatgtt cactggtaccagcaggttccaggaacagcccccaaactcctcatctatggtaacaacaatcggccctcgggggtccctgaccgattctctggctcc aagtctggcacctcagcctccctggccatcactgggctccagcctgaggatgaagttgattattactgccagtcctatgaccgcagcctgagtggtta tatcttcggaagtgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacc tgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 603)
>Herl36_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCARTPGY SSGWYSVWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVVTQPPSVSGAPGQRVTISCTGSSSN IGAGYD VHWYQQVPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAITGLQPEDEVD YYC QSYDRSLSGYIFGSGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKJ^WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 604)
>Herl37_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaaagatctgaacccttattcagtggtaactctcgg tatggacgtctggggcagagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcaca gtctgtgctgactcagccaccctcggtgtcagtggccccaggacaggcggccaggattccctgtgggggagacaacattggaagtaagagtgttc actggtaccagcagaggccaggccaggcccctgtcctggtcgtctttgatgatagtgaccggccctcagggatccctgagcgattctctggctcca attctgggcacacggccaccctgaccatcaacagggtcgaacccggggatgaggccgagtattattgtgaggtgtgggatggtggcgagagaca tgtggtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccag cacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac aaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggat gagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 605)
>Herl37_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLNP
YSVVTLGMDVWGRGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSVAPGQAARIPCG
GDNIGSKSVHWYQQRPGQAPVLWFDDSDRPSGIPERFSGSNSGHTATLTINRVEPGDEAEYY
CEVWDGGERHWFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK. (SEQ ID NO: 606)
>Herl38_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccataagctgggtccgccaggctccagggaaggggc tggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaa cacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagattcgagtagggtgggagcttatctggtgttt gactactggggccgggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtct gtgctgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatg atgtacactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacagcaaacgcccctcaggggtccctgaccgattctctgg ctccaagtctggcacctcagcctccctggccctcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagt ggttatgtcttcggaactgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccca gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaa caaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggga tgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 607)
>Herl38_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAISWVRQAPG KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSRV GAYLVFDYWGRGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSGAPGQRVTISCTGSS SNIGAGYDVHWYQQLPGTAPKLLIYGNSKRPSGVPDRFSGSKSGTSASLALTGLQAEDEADY YCQSYDSSLSGYVFGTGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKF^WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 608)
>Herl39_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacggcctgagagtcgaggacacggccgtgtattactgtgcgaaagaattggtcagtagagggagcctcacctttg actactggggcaaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctg tgttgacgcagccgccctcagtgtctggggccccagggcagggggtcaccatctcctgcactgggagcagctccaacatcggggcagattttgct gtacactggtaccaacaacttccagggacagcccccaaactcctcatcaatggtagcagccatcggccctcaggggtccctgaccgattctctggc tccaagtctggcccctcagcctccctggccatcactgggctccaagccgacgatgaggctgattatttttgccagtcctatgactacagactcaatgct ttagtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagc acctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg gtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagga gcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaaca aagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatg agctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccgg agaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 609)
>Herl39_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNGLRVEDTAVYYCAKELVS RGSLTFDYWGKGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSGAPGQGVTISCTGSSS NIGADF AVHWYQQLPGTAPKLLINGSSHRPSGVPDRFSGSKSGPSASLAITGLQADDEAD YFC QSYD YRLNALVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKE YKCKVSNKALP APBEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 610)
>Herl40_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagatagacggccgagggatgatgcttttgata tgtgggggagagggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacttaacatcc agatgacccagtctccctcttctgtgtctgcttctgttggagacagagtcagcatcacttgtcgggcgagtcagggaattggcagctggttattctggta tcagcagaaaccagggaaagcccctatcctcctgatgtccgctgtgtccggtttgcaaagtggagtcccatcacgattcagcggcagcggatctgg gacagatttcactctcacgatcagcagcgtacagcctgaggattttgcaacttactattgtcaacaggctcacagtttccctatcaccttcggccaagg gacacgactggagattaaacgtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtg gaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacg aagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccc atcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgct ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 611)
>Herl40_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRRP RDDAFDMWGRGTTVTVSSGGGGSGGGGSGGGGSALNIQMTQSPSSVSASVGDRVSΓΓCRAS QGIGSWLFWYQQKPGKAPILLMSAVSGLQSGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQ QAHSFPITFGQGTRLEIKRDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPE
VTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK. (SEQ ID NO: 612) >Herl41_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactccgcagactccgtgaagggccggttcaccatctccagagacaactccaag aacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgggaggtagagtgggatctacggcggcttttgat acatgggggcgagggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgt gctgactcagccgtcctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatg tacactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgcgtggt tatgtcttcggaactgggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagca cctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 613)
>Herl41_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYSADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGRVG STAAFDTWGRGTTVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSGAPGQRVTISCTGSSSN IGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYC QSYDSSLRGYVFGTGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 614)
>Herl42_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagatcgagccctagtgggagctactcgaactt ttggctactgggggcaggggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacag gctgtgctgactcagccgtcctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggtta tgatgtacactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtgacaccaatcggccctcaggggtccctgaccgattctct ggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctttgacagcagcctca gtggttcggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg gaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtct ccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc gggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggca gccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 615)
>Herl42_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARDRAL VGATRTFGYWGQGTTVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSGAPGQRVTISCTGS
SSNiGAGYD VHWYQQLPGTAPKLLIYGDTNRPSGVPDRFSGSKSGTSASLAΓΓGLQAEDEAD YYCQSFDSSLSGSVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKΈYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 616)
>Herl43_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagttgggtcgtttggtgattacaaagataaaag tggttacggcttctactttgactactggggccaaggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtg gcggaagtgcacagtctgtgctgactcagccaccctcggcgtctgggaccctcgggcagacggtcttcatctcttgttctggaagcagttccaacat cggaagtaattctgtgagttggtaccagcagctcccaggaacggcccccaaatttctcatttatcataataatcagcggccctcaggggtccctgagc gattctctggctccaagtctggcacctcagcctccctggccatccgtgggctccagtctgaggatgaggctgattactactgtgcatcttgggaggac agcctgaatggttgggtgttcggcggggggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccc accgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaag ccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagca atgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa tga (SEQ ID NO: 617)
>Herl43_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYΎCARVGSF GDYKDKSGYGFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTLGQTV FISCSGSSSNIGSNSVSWYQQLPGTAPKFLIYHNNQRPSGVPERFSGSKSGTSASLAIRGLQSED EAD YYCASWEDSLNGWVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKΓ^NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK. (SEQ ID NO: 618)
>Herl44_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgggaggtagagtgggatctacggcggcttttgata catggggccaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgt taacgcagccgccctcagtgtctggggccccagggcagagggtcgccatatcctgtacggggagcagctccaatattggggcaggttatgatgta cactggtttcagcaacttccaggaacagcccccaaactcctcatctttggtaacaagaatcggccctcaggggtccccgaccgattctctgcctctaa gtctggcaccgcagcctccctggccatcactgggctccaggctgaggatgagggtgattattactgccagtcctatgacagcagcctgagtggtgt gatcttcggcagagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcac ctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggt ggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgag ctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggag aacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggg gaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 619) >Herl44_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGRVG STAAFDTWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSGAPGQRV AISCTGSSS NIGAGYD VHWFQQLPGTAPKLLIFGNKNRPSGVPDRFSASKSGTAASLAITGLQAEDEGDYY CQSYDSSLSGVIFGRGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKΈYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ID NO: 620)
>Herl45_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgggagcggcggactacagtaattactttgacttttg gggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgctgac tcagccaccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatgtacact ggtaccagcaacttccaggagcagcccccaaactcctcatctatgggaacatcaatcggccctcaggggtccctgaccgattctctggctccaagt ctggcacctcagcctccctggccatcactgggctccaggctgaggatgagggtgattattactgccagtcctatgacagaagcctgagtgctaagct gttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg aactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtgga cgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagta caacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctg accaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 621)
>Herl45_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGAADY
SNYFDFWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSGAPGQRVTISCTGSSSNI
GAGYDVHWYQQLPGAAPKLLIYGNINRPSGVPDRFSGSKSGTSASLAITGLQAEDEGDYYCQ
SYDRSLSAKIFGGGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSWLFPPKPKDTLMISR
TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN
GKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNΉYTQ
KSLSLSPGK. (SEQ ID NO: 622)
>Herl46_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcggcgagtaatagttattactactttgactactggggc cagggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttcttctgagctgactca ggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagcctcagaaacttttatccaagttggtatcagcagaa gccaggacaggcccctgttcttgtcatttatggtaaaaatattcggccctcagggatcccagaccgattctctggctccggctcaggaagcacagctt ccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaaacatatgggggtggtattcggcg gagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctg ggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagc cacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagc ccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaag accacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctca tgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 623)
>Herl46_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNSY
YYFD YWGQGTLVTvssGGGGSGGGGSGGGGSALSSELTQDP AVSV ALGQTVRΓΓCQGDSLR
NFYPSWYQQKPGQAP VLVIYGKNIRPSGIPDRFSGSGSGSTASLTITGAQAEDEAD YYCNSRD
SSGKHMGWFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK. (SEQ ID NO: 624)
>Herl47_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acgcgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagatctgggaatagaccccctttggagtggtt attacacaccccttgactattggggccgggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcgg aagtgcactttcttctgagctgactcaggaccctgctctgtcggtggccttgggacagacagtcaggatcacatgtcaaggggacagcctcggagg ctttcatgcaagctggtaccaggagaagccaggacaggcccctgtatttgtcctctatggtaaaaacaaccggccctcagggatcccagaccgattc tctggctccacctcaggtaacacagctgccctgaccatcactggggctcaggcggaagatgaggctgactattactgtagctcccgggacagaagt ggtaaccatcgcgtcttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccacc gtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac atgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccg cgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaag gtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgg gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggt ggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 625)
>Herl47_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNALYLQMNSLRAEDTAVYYCARDLGI DPLWSGYYTPLDYWGRGTMVTVSSGGGGSGGGGSGGGGSALSSELTQDPALSVALGQTVRI TCQGDSLGGFHASWYQEKPGQAPVFVLYGKNNRPSGIPDRFSGSTSGNTAALTITGAQAEDE ADYYCS SRDRSGNHRVFGGGTKLTVLGDVREPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ ID NO: 626)
>Herl48_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaggggctacagtggaagttcctttgacgcctggg gccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgttgacgc agccgccatcagcgtccgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaatatcggaagtaagtctgtatactggtacc agcaactcccaggagcggcccccaaactcctcatctacaggaatagtcagcggccctcaggggtccctgaccgattctctgcctccaagtctggca cctctgcctccctggccatcagtgggctccggtccgaggatgaggctgactattactgtgcagcatgggatggcagcctgagtggacatttcttcgg aactgggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcct gggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgag ccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacag cacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga accaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaa gaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttct catgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 627)
>Herl48_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSG SSFDAWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTPGQRVTISCSGSSSNIGS KSVYWYQQLPGAAPKLLIYRNSQRPSGVPDRFSASKSGTSASLAISGLRSEDEAD YYCAAWD GSLSGHFFGTGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCXΛTVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK. (SEQ ID NO: 628)
>Herl49_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgggagaggcagcagagtggggacgatttggggaag ccttgacttttggggccaagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacag tctgtgctgacgcagccgccctcagtgtctgcggccccgggacagagggtcaccatctcctgctctggaaagagctccaacattggcggtaattct gtggcctggtaccagcaactcccgggaacagcccccaaagtcctcatttatgacaatgataagcgaccctcaggggttcctgaccgattctctggct ccaagtctggcacgtcagccaccctgggcatcaccggactgcagactggggacgaggccgattattactgcggatcctgggatagcagcctggg tgttgggatgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcc cagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgt ggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggga ggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctcc aacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgg gatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagc cggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcag caggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 629)
>Herl49_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGRGSRV GTIWGSLDFWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSAAPGQRVTISCSGK SSNIGGNSVAWYQQLPGTAPKVLIYDNDKRPSGVPDRFSGSKSGTSATLGITGLQTGDEADY YCGSWDSSLGVGMFGGGTK VTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQ YNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALP APΓEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 630)
>Herl50_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagatcgcgtttacgatttttggagtggttattata cgaggtacaactggttcgacccctgggggcgagggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggt ggcggaagtgcacaggctgtgctgactcagccgtcctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaa catcggaagtaattatgtatactggtaccagcagctcccaggaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccct gaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatggg atgacagcctgagtagtccggtgttcggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacaca tgcccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccct gaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaaga caaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtaca agtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccc tgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtggga gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccg ggtaaatga (SEQ ID NO: 631 )
>Her 150_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRVY DFWSGYYTRYNWFDPWGRGTTVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSASGTPGQRV TISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSE DEAD YYCAAWDDSLSSPVFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPP
KPKΏTLMISRTPEVTCVVVDVSHEDPEVKITSΓWYVDGVEVHNAKTKPREEQ YNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK. (SEQ ID NO: 632)
>Herl51_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccagactccagggaaggggc tggagtgggtctcagctattagtggtagtggtggtagcacatactacgcaaactccgtgaagggccggttcaccatctccagagacaattccaagaa cacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgcgggggggaggctacaacccttttgactcctggg gccaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgccctgactc agcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccggcagtgacgttggtggttataactatgtctcctggtacc aacagcacccaggcaaagcccccaaactcatgatttatgaggtcattaatcggccctcagggatttctaatcgcttctctggctccaagtctggcaac acggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcggctcatattcaagcagcagcactcttgtattcggcggag ggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggt ggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccac gaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcac gtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccc ccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaacc aggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 633)
>Herl51_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPG KGLEWVSAISGSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGGYN PFDSWGQGTMVTVSSGGGGSGGGGSGGGGSAQSALTQPASVSGSPGQSΠTSCTGTGSDVGG
YNYVSWYQQHPGKAPKLMIYEVINRPSGISNRFSGSKSGNTASLTISGLQ AEDEADYYCGSYS SSSTLVFGGGTKLWLGDVREPKSSDKTHTCPPCPAPELLGGPSWLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFTNfV\rn^DGVE\^HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK. (SEQ ID NO: 634) >Herl52_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagaggtcacaaaatgggatactttgactactggg gccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttcttctgagctgact caggaccctgctgtgtccgtggccttgggacagtcagtcaccatcacgtgtcggggagccagcctcagcaactattatgcaagctggtaccagcag aggccaggacaagcccctctacttgtcgtctctgataacaacatccggccctcagggatcccagaccgattctctggctccaggtcaggaaccaca gcttccttgagcatcactggggctcaggcggaagatgaggctgactattactgtcactcccgtgccagcagtgacacccatgtccgggtgtttggcg gcgggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctg ggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagc cacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagc ccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaa ccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaag accacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctca tgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 635)
>Herl52_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARGHKM GYFD YWGRGTLVTVSSGGGGSGGGGSGGGGSALSSELTQDP AVSV ALGQSVTITCRGASLS NYYASWYQQRPGQ APLLWSDNNIRPSGIPDRFSGSRSGTTASLSITGAQAEDEADYYCHSRA SSDTHVRVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVK-FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPEN^^i^KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK. (SEQ ID NO: 636)
>Herl53_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagggacagggtctctaactggaactactacggc caggacagctactttgactactggggccaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggc ggaagtgcacagtctgtgctgactcagccaccctccgcgtctggggcccccgggcagagggtcaccatttcttgttctgggaccaactccaacatc ggaagtaataatgtaaactggtatcagcaactcccaggaaaggcccccagactcctcatctacaataataatcagaggccctcaggggtccctgac cgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgagcttgaggctgattattattgttcagcatgggatgac agcctgcatggtccggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgccc accgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaag ccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagca atgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa tga (SEQ ID NO: 637)
>Herl53_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRVS
NWNYYGQDSYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGAPGQRVTI
SCSGTNSMGSNNVKWYQQLPGKAPRLLIYNNNQRPSGVPDRFSGSKSGTSASLAISGLQSEL
EAD YYCSAWDDSLHGPVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK. (SEQ ID NO: 638)
>Herl54_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagggacagggtctctaactggaactactacggc caggacagctactttggctactggggccaggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggc ggaagtgcactttcctatgagctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcagctccaac atcggaagtaatactgtaacctggtaccagcagctcccaggaacggccccccaactcctcttccataataatgaccagcggccctcaggggtccct gaccgattctctggctccaagtctggcacctcaggctccctggccatcagtgggctgcagtctgaggatgaggctgattattactgttcagcatggga tgacggcctgaatgctgtaatattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatg cccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctga ggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca aagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaag tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggaga gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaatga (SEQ ID NO: 639)
>Herl54_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARDRVS
NWNYYGQDSYFGYWGQGTMVTVSSGGGGSGGGGSGGGGSALSYELTQPPSASGTPGQRVT
ISCSGSSSNIGSNTVTWYQQLPGTAPQLLFHNNDQRPSGVPDRFSGSKSGTSGSLAISGLQSED
EADYYCSAWDDGLNAVIFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK. (SEQ ID NO: 640)
>Herl56_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtgcagtctggggcagaggtgaaa aagcccggggagtctctgaagatctcctgtaagggttctgggtacagctttagtaactactggatcggctgggtgcgccagatgcccgggaaaggc ctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatca gtaccgcctacctgcagtggagcagcctgaaggcctcggacagtgccatgtattactgtgcgagacatgattcgactatgggatatgatgcttttcat atgtggggccaaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtg ctgactcagccgtcctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcggggcaggttatgatgt acactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggctcc aagtctggcacctcagcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacagcagcctgagtggc cctgtggtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgccc agcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggag gagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaa caaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggga tgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 641) >Herl56_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMP GKGLEWMGHYPGDSDTRYSPSFQGQ VTISADKSISTAYLQWSSLKASDSAMYYCARHDSTM GYDAFHMWGQGTLVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSGAPGQRVTISCTGSSS NIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAJTGLQAEDEADYY CQSYDSSLSGPVVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 642)
>Herl57_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagataattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagtgggggccggggagaactactaccacta ctacatcatggacgtctggggccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaag tgcactttcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagagagtcagggtcacatgccaaggagacagcctcagaggctat tatgcaagctggtaccagcagaagccaggacaggcccctgttcttgtcatctatggtgaaaacaaccggccctcagggatcccagaccgattctct ggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccggcacagcagtggt aattatctggtgttcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg gaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtct ccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc gggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggca gccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 643)
>Herl57_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSMSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGAG ENYYHYYIMDVWGRGTLVTVSSGGGGSGGGGSGGGGSALSSELTQDPAVSVALGQRVRVT CQGDSLRGYY ASWYQQKPGQAPVLVIYGENNRPSGIPDRFSGSSSGNTASLTITGAQAEDEA DYYCNSRHSSGNYLVFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALP APEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ BD NO: 644)
>Herl58_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagaactacggcagatgcttttgatatctggggca gagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttcttctgagctgactcag gaccctgctgtgtctgtggccttgggacagacagtcaagatcacatgccaaggcgacagcctcagaaactattatgcaggctggtaccagcagaa gccaggacaggcccctgtacttgtcatctatggtgaaaacaagcggccctcagggatccctgaccgattctctggctccaactcaggaaacacagc ttccttgaccctcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtagtaacctcgtggtattcggcggagg gaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtg gaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacg aagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgt accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccc atcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag gtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgct ccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 645)
>Herl58_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARTTAD AFDIWGRGTMVTVSSGGGGSGGGGSGGGGSALSSELTQDP AVSV ALGQTVKITCQGDSLRN YYAGWYQQKPGQAPVLVIYGENKRPSGIPDRFSGSNSGNTASLTLTGAQAEDEADYYCNSR DSSSNLVVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFTsTWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGK. (SEQ ED NO: 646)
>Herl59_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctataccatgagctgggtccgccaggctccagggaaggggc tggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaa cacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcaaggaatagcagcaattggagtggtgcttttgatatc tgggggcgggggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgct gacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcattggaacccactcaaacatcggggcaggttacgctgtg aactggtaccagcagcttccaggaacagcccccaaactcctcatctatggtaataacaatcggccctcaggggtccctgaccgattctctggctcca agtctggcacctcagcctccctggccatcaatgggctccaggctgacgatgagtctgattattattgccagtcctatgacgccagtctgagagttttatt cggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaa ctcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 647)
>Her 159_Protein_leader-stop
MEAP AQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPG
KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNSSNW
SGAFDΓWGRGTTVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSGAPGQRVTISCIGTHSNIG
AGYAVNWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAINGLQADDESDYYCQS
YD ASLRVLFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKJTSΓWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK. (SEQ ID NO: 648)
>Herl60_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagagatgacgatttttggagtggttatccattcctc tactactactacggtatggacgtctggggccgagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtg gcggaagtgcacagtctgtcgtgacgcagccgccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaactagttccaaca tcggaagtaatgctgtaaactggtaccagcaactcccaggaacggcccccaaactcctcatctataataataatcagcggccctcaggggtccctga ccgattctctggctccaagtctggcacctcagcctccctggccatcagtggactccagtctgaggatgaggctgattattactgtgcagcatgggatg acagcctgaatgtttatgtggtattcggcggagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatg cccaccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctga ggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca aagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaag tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggaga gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaatga (SEQ ID NO: 649)
>Her 160_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDDF WSGYPFLYYYYGMDVWGRGTMVTVSSGGGGSGGGGSGGGGSAQSVVTQPPSASGTPGQR VTISCSGTSSNIGSNAVNWYQQLPGTAPKLLIYNNNQRPSGVPDRFSGSKSGTSASLAISGLQS EDEAD YYCAAWDDSLNVYWFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK. (SEQ ID NO: 650)
>Herl61_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacttactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgctaaatggggctgggattactatgacacaactggtc atgatgcctttgatttctggggccgggggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagt gcacaggctgtgctgactcagccgtcctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcgggg cagattattatgtaaattggtatcagcaacttccaggaaaagcccccgaaatcgtaatttttaatgatgacaatcggccctcaggggtccctaaccgatt ctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaagatgaggctgattattattgccagtcttatgacagtgtcct gagtgcttatgtcttcggaactgggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc gtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg gaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtct ccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc gggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggca gccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 651)
>Herl61_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGW DYYDTTGHDAFDFWGRGTMVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSVSGAPGQRVTIS CTGSSSNIGAD YYVNWYQQLPGKAPEIVIFNDDNRPSGVPNRFSGSKSGTSASLAITGLQ AED EADYYCQSYDSVLSAYVFGTGTKVTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK. (SEQ ID NO: 652)
>Herl62_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctggtggagactggggctgaggtgaag aagcctggggcctcaatgaaggtctcctgcaaggcttctggatacagcttcaccgactactatatgcactgggtgcgacaggcccctggacaaggg cttgagtggatgggatggatcaaccctaatagtggtgacacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatc accacagcctacatggagctgagcaggctgagatctgacgacacggccgtatattactgtgcgacagagaggtataacagtggctgggaatggg gccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgccctgactc agcctgcctccgtgtctgggtctcctggacagtcgatcaccgtctcctgcactggaaccagcagtgacgttggtgcttataactatgtctcctggtacc aacaacacccaggcaaagcccccaaactcatgatttatgatgtcactactcggccctcaggggtttctaatcgcttctctggctccaagtctggcaac acggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcacctcatatactcgcagcagcactgtggtcttcggcggag ggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggt ggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccac gaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcac gtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccc ccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaacc aggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg ctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 653)
>Her 162_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLVETGAEVKKPGASMKVSCKASGYSFTDYYMHWVRQA
PGQGLEWMGWINPNSGDTNYAQKFQGRVTMTRDTSITTAYMELSRLRSDDTAVYYCATER
YNSGWEWGRGTLVTVSSGGGGSGGGGSGGGGSAQSALTQPASVSGSPGQSITVSCTGTSSD
VGAYNYVSWYQQHPGKAPKLMIYDVTTRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYY
CTSYTRSSTWFGGGTKLTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK. (SEQ ID NO: 654)
>Herl63_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcggtccatggctacggagactccgtggatgatgctc ttgatgtctggggccgaggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtc tgtgctgacgcagccgccctcagcgtctgggacccccgggcagacgatctccatctcttgttctggaagcaactccaacatcggaacttatagtgtta gctggtaccagcagctcccacgagcggcccccagactcctcgtctatgctaatgatcgccggccctcaggggtccctgaccgattctctggctcca agtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagtatgggatgacaggttgaatggttttg tcttcggaactgggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg aactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtgga cgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagta caacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctg accaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID
NO: 655)
>Herl63_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVHGYG DSVDDALDVWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSASGTPGQTISISCSGSN SNIGTYSVSWYQQLPRAAPRLLVYANDRRPSGVPDRFSGSKSGTSASLAISGLQSEDEAD YYC AVWDDRLNGFVFGTGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ED NO: 656) >Herl64_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgaagtatggtggctacgacgctgatgcctttgatgt ctggggccgagggacaatggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtcgt gacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctgctctggaagtagctccaacattggagattattatgtatcctgg taccagcaactcccaggaacggcccccacactcctcatttatgacaatgataagcgaccctcagaagttcctgaccgattctctggctccaagtctgg cacgtcggccaccctcggcatcaccggactccagactggggacgaggccgattattactgcacttcatgggatagcagcctgagtgctggggtgtt cggcggagggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaa ctcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggac gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaa ctacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ED NO: 657)
>Her 164_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYGGY DADAFDVWGRGTMVTVSSGGGGSGGGGSGGGGSAQSVVTQPPSVSAAPGQKVTISCSGSSS NIGDYYVSWYQQLPGTAPTLLIYDNDKRPSEVPDRFSGSKSGTSATLGITGLQTGDEADYYCT SWDSSLSAGVFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK. (SEQ ED NO: 658)
>Herl65_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtaaggtgcagctggtgcagtctgggtctgagttgaaga agcctggggcctcagtgaaggtttcctgccaggcttctggatacaccatcactaaccatagcatgaattgggtgcgacaggcccctgggcaaggg cttgagtggatgggatggatcaacaccaacactgggaaccctacgtatgcccagggcttcacaggacggtttgtcttctccttggacacctctgcca acacggcaactttgcagatcaccaacgtgcaggctgaggacacagccgtctactactgtgcgagagaggggagtatagacgtgtctggaacgcc ctactactacggaatggacgcctgggggcaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggaggtggcagcggcggtg gcggatcgcagtctgtgctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttgg tggttataactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatgagggcagtgagcggccctcaggggttcctaat cgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaac caggagcactcgagttttcggcggagggaccaagctgaccatcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccac cgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtca catgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagcc gcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaa ggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccc atcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaat gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagca ggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaat ga (SEQ ED NO: 659)
>Herl65_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGKVQLVQSGSELKKPGASVKVSCQASGYTITNHSMNWVRQAP GQGLEWMGWENTNTGNPTYAQGFTGRFVFSLDTSANTATLQITNVQAEDTA VYYCAREGSI DVSGTP YYYGMDAWGQGTTVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSmSCT GTSSDVGGYNYVSWYQQHPGKAPKLMEYEGSERPSGVPNRFSGSKSGNTASLTISGLQAEDE ADYYCSSYTTRSTRVFGGGTKLTILGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKJ^SI^^^yrVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK. (SEQ ID NO: 660)
>Herl66_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctggtgcagtctggggcagaaataaaa aagccgggggagtctctgaagatctcctgtgagggttctggatacaggtttaccagccactggatcggctgggtgcgccagatgcccgggaaagg cctggagtggatggggatcatctatcctggtgactctgataccagatacagcccgtccttccaaggccaggtcaccatctcagccgacaagtccatc agcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagacatagtgcgacgcatgatgcttttgatatct ggggccggggcaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacagtctgtgttga cgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgtagtgggagcagctccaacatcgggacaggttacgatgttcac tggtaccagcaacttccaggaacagcccccaaactcctcatctatagtttcaataagcggccctcaggggtccctgaccggttctctgcctccaagtc tggcacctcagcctccctggtcatcactgggctccaggctgaggatgaggctgattattactgccagtcctatgacaatttgagtggtccccatgtggt tttcggcacagggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg aactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtgga cgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagta caacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctg accaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcagggga acgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 661)
>Herl66_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLVQSGAEDCKPGESLKISCEGSGYRFTSHWIGWVRQMPG
KGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM YYCARHSATHD
AFDIWGRGTLVTVSSGGGGSGGGGSGGGGSAQSVLTQPPSVSGAPGQRVTISCSGSSSNIGTG
YDVHWYQQLPGTAPKLLIYSFNKRPSGVPDRFSASKSGTSASLVITGLQAEDEADYYCQSYD
NLSGPHVVFGTGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK. (SEQ ID NO: 662)
>Herl67_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtcaggtgcagctgcaggagtcgggcccaggactggtg aagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcaccagtgatctttcctactggggctggctccgccagccccccggga agggtctggagtggattgcgagtggtggtgacggtgagagcacctactacaacccgtccctcaacggtcgagtcaccttttccgtggacacgccca agaaccaattctccctgaggctgagctctgtgaccgccgcagacacggctgtatattactgtgcgagacacccactctactattgtagtggtggtcgc tgctactccgggaactttgacttttggggccagggaaccctggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtgg cggaagtgcacaggctgtgctgactcagccgtcctcagcgtctgggacccccggtcagagggtcaccatttcttgttctggaacgacccccaatatt ggaagtaattttgtctactggtatcaacaactcccagggacggcccccaaactcctcatctacaggaatgagcagcgcccttcaggggtccctgtcc gattctctggctccaagtctggcacatcagcctccctggccatcagtgacctccggtccgaggatgaggctgactattactgtgcagcgtgggatga cagcctgagtggtgtggtcttcggcggggggaccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcc caccgtgcccagcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaa gccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcc cccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagc aatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagag caggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaa atga (SEQ ID NO: 663) >Her 167_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGQVQLQESGPGLVKPSETLSLTCTVSGGSITSDLSYWGWLRQP PGKGLEWIASGGDGESTYYNPSLNGRVTFSVDTPKNQFSLRLSSVTAADTA VYYCARHPLYY CSGGRCYSGNFDFWGQGTLVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSASGTPGQRVTIS CSGTTPNIGSNFVYWYQQLPGTAPKLLIYRNEQRPSGVP VRFSGSKSGTSASLAISDLRSEDEA DYYCAAWDDSLSGWFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKLFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPmKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK. (SEQ ID NO: 664)
>Herl68_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaggggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcaacttcgttgggttacggtgactttgactactgggg gcgagggaccacggttaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcacaggctgtgctgactca gccgtcctcagcgtctggggcccccgggcacagggtcatcatctcttgttctggaagcagctccaacatcggaagttattatgtaagctggtaccag cagctcccaggagcggcccccaaactcctcatctatcgtaatgatgagcggccctcaggggtccctgcccgattctctggctccacgtctggcacct cagcctccctggccatcagtgggctccactctgaggatgaggctgattattattgtgcagcatgggatgacagcctgaatggtccggttttcggcgg agggaccaaggtcaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgg gtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcc acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagca cgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcc cccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaac caggtcagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcat gctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 665)
>Herl 68_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATSLGY GDFDYWGRGTTVTVSSGGGGSGGGGSGGGGSAQAVLTQPSSASGAPGHRVIISCSGSSSNIGS YYVSWYQQLPGAAPKLLIYRNDERPSGVPARFSGSTSGTSASLAISGLHSEDEAD YYCAAWD DSLNGPVFGGGTKVTVLGDVREPKSSDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK. (SEQ ID NO: 666)
>Herl69_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagaggctttggtgactactggggccgggggaca atggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttcttctgagctgactcaggaccctgct gtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagcctcagaagttattatgcaagctggtaccagcagaagccaggaca ggcccctgtacttgtcatctatgctaaaaacaaccgaccctcagggatcccagaccgattctctggctccgactcaggaaacacagcttccttgacca tcactggggctcaggcggaagatgaggctgactattactgtctctcccgggacagcagtggtaaccatctggtattcggcggagggaccaagctg accgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtggaccgtcagtc ttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctga ggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggt cagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctg acctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatg catgaggctctgcacaaccactacacgcagaagagcctctccccgtctccgggtaaatga (SEQ ID NO: 667)
>Herl69_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARGFGD YWGRGTMVTVSSGGGGSGGGGSGGGGSALSSELTQDPAVSVALGQTVRITCQGDSLRSYYA SWYQQKPGQ AP VLVIYAKNNRPSGIPDRFSGSDSGNTASLTITGAQAEDEAD YYCLSRDSSG NHLVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPSP GK. (SEQ ID NO: 668)
>Herl70_CDS atggaagcaccagcgcagcttctcttcctcctgctactctggctcccagataccaccggtgaggtgcagctgttggagtctgggggaggcttggtac agcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagaactacggcagatgcttttgatatctggggga gggggaccacggtcaccgtctcgagtggaggcggcggttcaggcggaggtggctctggcggtggcggaagtgcactttcttctgagctgactca ggaccctgctgtgtctgtggccctgggacagacagtcagcatcacatgccagggagacagcctcagaaacttttatgcaagctggtacctgcagaa gccaggacaggccccaatacttgtcatctatggtaaaaacaagcggccctctgggatcccagaccgagtctctggctccagctcagaagacacag cttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtggtaacgtggtcttcggcgggggga ccaagctgaccgtcctaggtgacgtacgcgagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggtgga ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtac cgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccat cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggt cagcctgacctgcctggtcaaaggcttctatccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac gcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctc cgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccccgtctccgggtaaatga (SEQ ID NO: 669)
>Herl70_Protein_leader-stop
MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTTAD AFDIWGRGTTVTVSSGGGGSGGGGSGGGGSALSSELTQDP AVSV ALGQTVSITCQGDSLRNF YASWYLQKPGQ APILVIYGKNKRPSGIPDRVSGSSSEDTASLTITGAQAEDEAD YYCNSRDSS GNVVFGGGTKLTVLGDVREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPS PGK. (SEQ ID NO: 670)
> Stumpy Peptide: Her2 N-terminal membrane proximal region of p95 (amino acid 645-656 of HER2) fused with EKK sequence
EQRASPLTSπS-EKK (SEQ ID NO: 671)
> Scrambled Peptide: Scrambled sequence, used as a negative control/de-selection protein
PEISLSQRITAS-EKK (SEQ ID NO: 672)

Claims

CLAIMSWe claim:
1. A binding protein that specifically binds ErbB2, wherein the binding protein is an ErbB2 agonist.
2. The binding protein of claim 1 which reduces cellular proliferation in an ErbB2- expressing cancer cell.
3. The binding protein of claim 1 or claim 2 which increases apoptosis in an ErbB2- expressing tumor.
4. The binding protein of claim 1 which reduces the growth of an ErbB2-expressing tumor.
5. The binding protein of claim 2 wherein the ErbB2-expressing cancer cell is a breast cancer cell.
6. The binding protein of claim 2 wherein the ErbB2 expressing cancer cell is from a cell line selected from the group consisting of: SKBR3, BT474, MDA-MB-453 and MDA-MB-361.
7. A binding protein that specifically binds ErbB2, wherein the binding protein preferentially binds an ErbB2 extracellular domain (ECD) homo-dimer over ErbB2 ECD monomer and shed ErbB2 ECD.
8. The binding protein of claim 1 , wherein the binding protein preferentially binds an ErbB2 extracellular domain (ECD) homo-dimer over ErbB2 ECD monomer and shed ErbB2 ECD.
9. The binding protein of claim 2 or claim 7, that possessess one or more or the following properties:
(a) increases ErbB2 phosphorylation in a breast cancer cell;
(b) increases the phosphorylation of one or more of AKT, MAPK, MEK, ERK1 and ERK2; (c) binds ErbB2 ECD in a location selected from the group consisting of: the L1/CR1 domain, the L2 domain, the CR2 domain;
(d) internalizes in an ErbB2-expressing cell;
(e) decreases ErbB2 ECD shedding compared to cells not treated with the binding protein;
(f) decreases the amount of cell surface ErbB2 compared to cells not treated with the binding protein;
(g) binds macaque ErbB2; (h) binds mouse ErbB2; (i) has Fc dependent cellular cytotoxicity activity with ErbB2-expressing cells;
(i) is stable in plasma for at least 96 hours;
(k) has antiproliferative activity that is reversible by inhbiting MEK-mediated phosphorylation, ERK2-mediated phosphorylation, or both; or
(I) enhances the cytotoxic effect of a chemotherapeutic.
10. The binding protein of claim 1 which is an antibody, an antigen-binding fragment of an antibody or a small modular immunopharmaceutical (SMIP).
11. The binding protein of claim 10 which is an antigen-binding fragment of an antibody, wherein the antigen-binding fragment is selected from the group consisting of: a Fab fragment, an F(ab')2 fragment, an scFv, a dAb, and Fv fragment and a VHH.
12. The binding protein of claim 1 , which is human antibody or an antigen-binding fragment thereof.
13. The binding protein of claim 1 , wherein the ErbB2 is human ErbB2 (SEQ ID NO: 246).
14. A binding protein that specifically binds ErbB2, wherein the binding protein comprises:
(a) a VH domain or an antigen-binding portion thereof comprising the CDR1 ,
CDR2 and CDR3 amino acid sequences set forth in any one of SEQ ID NOS: 1 , 3, 5, 7, 9,
11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 , 53, 55, 57,
59, 61 , 65, 67, 251 , 255, 259, 263, 267, 271 , 275, 279, 283, 287, 291 , 295, 299, 303, 307, 311 , 315, 319, 323, 327, 331 , 335, 339, 343, 347, 351 , 355, 359, 363, 367, 371 , 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483, 487, 491, 495, 499, 503, 507, 511, 515, 519 and 523; or
(b) a VL domain or an antigen-binding portion thereof comprising the CDR1 ,
CDR2 and CDR3 amino acid sequences set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521 and 525; or
(c) a VH domain or an antigen-binding portion thereof of (a) and a VL domain or an antigen-binding portion thereof of (b).
15. The binding protein of claim 14, comprising the VH CDR1, CDR2 and CDR3 amino acid sequences and the VL CDR1, CDR2 and CDR3 sequences of any one of: S1R2A_CS_1F7, S1R2A_CS_1D11, S1R2C_CS_1D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3, S1R3B2_DP47_1E8,
S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1R2A_CS_5A1, S1 R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9, S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1R2A_CS_7D4, S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1E11, S5R2_DP47_1H11, S6R3_CS_1G5, S6R2_DP47_1H11, S5R3_DP47_1A10, S5R2_DP47_1D11, S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1 C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1 H9..
16. A binding protein that specifically binds ErbB2, wherein the binding protein comprises:
(a) a VH domain having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 65, 67, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327,
331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483, 487, 491, 495, 499, 503, 507, 511, 515, 519 and 523; or
(b) a VL domain having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301,
305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521 and 525; or (c) a VH domain of (a) and a VL domain of (b); or
(d) VH domain and VL domain amino acid sequence that are at least 90% identical to the VH and VL, amino acid sequences, respectively, in any one of S1R2A_CS_1F7, S1R2A_CS_1D11, S1R2C_CS_1D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3, S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1 R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1R2A_CS_5A1, S1R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9,
S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1R2A_CS_7D4, S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1E11, S5R2_DP47_1H11, S6R3_CS_1G5, S6R2_DP47_1 H11 , S5R3_DP47_1 A10, S5R2_DP47_1 D11 , S5R2_CS_1A8,
S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1 H9.
17. The binding protein of claim 16, wherein the binding protein comprises: (a) a VH domain having an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 65, 67, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483, 487, 491, 495, 499, 503, 507, 511, 515, 519 and 523; or
(b) a VL domain having an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485, 489, 493, 497, 501, 505, 509, 513, 517, 521 and 525; or
(c) a VH domain of (a) and a VL domain of (b); or
(d) VH and VL amino acid sequences that are at least 95% identical to the VH and VL amino acid sequences, respectively, in any one of S1R2A_CS_1F7, S1R2A_CS_1D11, S1R2C_CS_1D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1 R3C1_CS_1 D3, S1 R3B2_DP47_1 E8, S1 R3B2_BMV_1 G2, S1 R3B2_BMV_1 H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1,
S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6, S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8,
S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1R3A1_CS_9C1, S1R2A_CS_5A1, S1R2A_CS_8C8, S1R3A1_CS_13H5, S1R2B_CS_5E9, S1R3A1_CS_8F9, S1R3A1_CS_14B5, S1R2A_CS_9E10, S1R3A1_CS_7A10, S1R3A1_BMV_6H7, S1R3A1_CS_12A11, S1R3A1_CS_13D12, S1R3A1_CS_7A8, S1R2A_CS_2C9, S1R3A1_CS_12D1, S1R2A_CS_7D4, S1R3A1_CS_15B8, S6R3_DP47_1A10, S6R2_DP47_1 E11 , S5R2_DP47_1 H11 , S6R3_CS_1 G5, S6R2_DP47_1 H11 ,
S5R3_DP47_1A10, S5R2_DP47_1D11, S5R2_CS_1A8, S6R3_CS_1B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1B10, S6R3_DP47_1C12, S5R2_DP47_1D10, and S6R3_DP47_1H9.
18. The binding protein of claim 16, wherein the binding protein comprises: (a) a VH domain having the amino acid sequence of any one of SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 65 or 67, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 427, 431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483, 487, 491, 495, 499, 503, 507, 511, 515, 519 and 523; or
(b) a VL domain having the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 63, 64, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441 , 445, 449, 453, 457, 461 , 465, 469, 473, 477, 481 , 485, 489, 493, 497, 501 , 505, 509, 513, 517, 521 and 525; or
(c) a VH domain of (a) and a VL domain of (b); or
(d) VH and VL amino acid sequences of the VH and VL amino acid sequences, respectively, in any one of S1 R2A_CS_1 F7, S1 R2A_CS_1 D11 , S1 R2C_CS_1 D3, S1R2C_CS_1H12, S1R2A_CS_1D3, S1R3B2_BMV_1E1, S1R3C1_CS_1D3,
S1R3B2_DP47_1E8, S1R3B2_BMV_1G2, S1R3B2_BMV_1H5, S1R3C1_CS_1A6, S1R3B2_DP47_1C9, S1R3B2_DP47_1E10, S1R3C1_CS_1B10, S1R3A1_BMV_1F3, S1R3B1_BMV_1G11, S1R3A1_BMV_1G4, S1R3B1_BMV_1H11, S1R3A1_CS_1B9, S1R3B1_BMV_1H9, S1R3A1_CS_1B10, S1R3B1_BMV_1C12, S1R3C1_BMV_1H11, S1R3B1_BMV_1A10, S1R3A1_CS_1D11, S1R3C1_DP47_1H1, S1R3A1_CS_1B12, S1R3B1_BMV_1H5, S1R3A1_DP47_1A6, S1R3B1_DP47_1E1, S1R3B1_BMV_1A1, S1R3B1_DP47_3A2, S1R3A1_DP47_11B7, S1R3A1_DP47_11D1, S1R3A1_DP47_7F3, S1R2B_DP47_4E3, S1R3C1_DP47_2G2, S1R3A1_DP47_11H6, S1R3A1_BMV_3B1, S1R3A1_DP47_6B9, S1R2A_CS_10B8, S1R3A1_DP47_7A6, S1R3B2_DP47_2G3, S1R2B_CS_6H11, S1R3A1_DP47_10G1, S1R3A1_DP47_7C1, S1R2A_DP47_5D6,
S1R3A1_DP47_11F6, S1R3A1_DP47_11D3, S1R3A1_CS_8A8, S1R3A1_BMV_5D10, S1R3A1_DP47_11C1, S1R3A1_DP47_4E1, S1R3A1_DP47_10E1, S1R3A1_CS_11C3, S1R3A1_CS_13H11, S1R3A1_CS_2D9, S1R2A_CS_3D4, S1R3A1_DP47_2H6, S1R3A1_DP47_4G1, S1R2A_DP47_3C1, S1R3A1_DP47_7B2, S1R3B2_DP47_4E2, S1R3A1_CS_16C2, S1R3A1_CS_11E5, S1R3A1_CS_16D7, S1R2A_CS_10B10, S1R3A1_CS_15C2, S1 R3A1_CS_9C1 , S1R2A_CS_5A1 , S1 R2A_CS_8C8, S1 R3A1_CS_13H5, S1 R2B_CS_5E9, S1 R3A1_CS_8F9, S1 R3A1_CS_14B5, S1 R2A_CS_9E10, S1R3A1_CS_7A10, S1 R3A1_BMV_6H7, S1 R3A1_CS_12A11 , S1 R3A1_CS_13D12, S1 R3A1_CS_7A8, S1 R2A_CS_2C9, S1R3A1_CS_12D1 , S1 R2A_CS_7D4, S1 R3A1_CS_15B8, S6R3_DP47_1 A10, S6R2_DP47_1 E11 , S5R2_DP47_1 H11 , S6R3_CS_1G5, S6R2_DP47_1 H11 , S5R3_DP47_1A10, S5R2_DP47_1 D11 , S5R2_CS_1A8, S6R3_CS_1 B7, S6R2_CS_1E5, S6R3_BMV_1C2, S5R2_DP47_1 B10, S6R3_DP47_1C12, S5R2_DP47_1 D10, and S6R3_DP47_1 H9.
19. The binding protein of any one of claims 9 or 14-18, which is a SMIP.
20. A SMIP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOS: 159, 173, 175, 177, 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211 , 213, 215, 217, 219, 221 , 223, 225, 227, 229, 231 , 233, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668 and 670, excluding the leader sequence.
21. The SMIP of claim 20, comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOS: 159, 173, 175, 177, 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211 , 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668 and 670, excluding the leader sequence.
22. The SMIP of claim 20, comprising the amino acid sequence of any one of SEQ ID NOS: 159, 173, 175, 177, 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211 , 213, 215, 217, 219, 221 , 223, 225, 227, 229, 231 , 233, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668 and 670, excluding the leader sequence.
23. The human monoclonal antibody or antigen-binding portion according to paragraph 1 or a SMIP according to any one of claims 20-22, that specifically binds a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO. 242;
(b) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO. 243;
(c) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO. 244;
(d) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO. 245; and (e) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO.
671.
24. The human monoclonal antibody or antigen-binding portion according to paragraph 1 or a SMIP according to any one of claims 20-22, that specifically binds ERBB2 in a region selected from the group consisting of:
(a) a region of the extracellular domain comprising the amino acid sequence ASPLTS;
(b) a region of the extracellular domain comprising the amino acid sequence EQRASPLTS; and (c) a region of the extracellular domain comprising the amino acid sequence
EQRASPLTSIIS.
25. A nucleic acid molecule encoding the binding protein of any one of claims 14-18 or encoding the SMIP of any one of claims 20-24.
26. A nucleic acid molecule that encodes a binding protein that specifically binds ErbB2, wherein the nucleic acid molecule comprises a nucleotide sequence selected from: (a) the nucleotide sequence of any one of SEQ ID NOS: 96, 98, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156; 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, 428, 432, 436, 440, 444, 448, 452, 456, 460, 464, 468, 472, 476, 480, 484, 488, 492, 496, 500, 504, 508, 512, 516, 520 or 524; or
(b) the nucleotide sequence of any one of SEQ ID NOS: 97, 99, 101 , 103, 105, 107,
109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 ,133, 135, 137, 139, 141 , 143, 145, 147, 149, 151 , 153, 155, 157; 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294,
298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458, 462, 466, 470, 474, 478, 482, 486, 490, 494, 498, 502, 506, 510, 514, 518, 522, or 526; or (c) both the nucleotide sequence of (a) and the nucleotide sequence of (b).
27. The nucleic acid molecule of claim 26, comprising the nucleotide sequence of any one of SEQ ID NOS: 158, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,r 232, 533, 535, 537, 539, 541 , 543, 545, 547, 549, 551 , 553, 555, 557, 559, 561 , 563, 565, 657, 569, 571 , 573, 575, 577, 579, 581 , 583, 585, 587, 589, 591 , 593, 595, 597, 599, 601 , 603, 605, 607, 609, 611 , 613, 615, 617, 619, 621 , 623, 625, 627, 629, 631 , 633, 635, 637, 639, 641 , 643, 645, 647, 649, 651 , 653, 655, 657, 659, 661 , 663, 665, 667 and 669.
28. A composition comprising a binding protein of any one of claims 1-18, 23 or 24 or the SMIP of any one of claims 20-22.
29. The composition of claim 28, further comprising an additional therapeutic or diagnostic agent.
30. The composition of claim 28, comprising two or more binding proteins according to any one of claims 1-18, 23 or 24 or SMIPS according to any one of claims 20-220
31. The composition of claim 30, wherein the two or more binding proteins or SMIPS do not cross-block each other for binding to ErbB2.
32. The composition of claim 29 that comprises an additional therapeutic agent, wherein the therapeutic agent is a chemotherapeutic or anti-inflammatory agent.
33. A host cell comprising a nucleic acid molecule of any one of claims 25-27.
34. The host cell of claim 33 selected from the group consisting of an HEK cell, an NSO cell and a CHO cell.
35. A method for producing a binding molecule of claim 1 or a SMIP of claim 20, comprising the step of culturing the host cell of claim 33 under conditions the permit protein expression.
36. A method for reducing ErbB2-mediated proliferation of a cancer cell comprising the step of administering to a subject or mammal in need thereof an effective amount of a binding protein of claim 2 or a composition of claim 28.
37. A method for reducing tumor growth of an ErbB2-expressing tumor, comprising administering to a subject or mammal in need thereof an effective amount of a binding protein of claim 2 or a composition of claim 28.
38. A method for increasing apoptosis in an ErbB2-expressing tumor, comprising administering to a subject or mammal in need thereof an effective amount of a binding protein of claim 2 or a composition of claim 28.
39. The method of claim 36 or 37, further comprising administering a chemotherapeutic agent.
40. The method of claim 39, wherein the chemotherapeutic agent is selected from the group consisting of: Taxol, Doxorubicin, Gemcitabine and Cisplatin.
41. A method for reducing ErbB2 ectodomain shedding in an ErbB2-expressing cell comprising the step of contacting the cell with a binding protein of claim 1 or a SMIP of claim 20.
42. A method for reducing the amount of cell surface ErbB2 in a cell comprising the step of contacting the cell with a binding protein of claim 1 or a SMIP of claim 20.
43. The binding protein of claim 1 , which is detectably labeled.
44. A method for detecting an ErbB2 expressing tumor in a subject, comprising administering the binding protein of claim 43.
45. A method for detecting ErbB2 in a sample from a subject comprising the step of contacting the sample with a binding protein of any one of claims 14-19 or a SMIP of claim
20-22 under conditions that permit binding and detecting binding, wherein binding indicates the presence of ErbB2.
46. A method of treating cancer characterized by ErbB2 expression comprising administering to a mammal or subject in need thereof an effective amount of a binding protein of any of claims 1-19 or a SMIP of any one of claims 20-22.
PCT/US2008/012212 2007-10-25 2008-10-27 Erbb2 binding proteins and use thereof WO2009055074A2 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US51107P 2007-10-25 2007-10-25
US61/000,511 2007-10-25
US6243308P 2008-01-24 2008-01-24
US61/062,433 2008-01-24
USPCT/US2008/006905 2008-05-29
PCT/US2008/006905 WO2008150485A2 (en) 2007-05-29 2008-05-29 Erbb2 binding proteins and use thereof

Publications (3)

Publication Number Publication Date
WO2009055074A2 true WO2009055074A2 (en) 2009-04-30
WO2009055074A8 WO2009055074A8 (en) 2009-08-06
WO2009055074A3 WO2009055074A3 (en) 2009-09-11

Family

ID=40512895

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/012212 WO2009055074A2 (en) 2007-10-25 2008-10-27 Erbb2 binding proteins and use thereof

Country Status (1)

Country Link
WO (1) WO2009055074A2 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011147986A1 (en) * 2010-05-27 2011-12-01 Genmab A/S Monoclonal antibodies against her2
US9107862B2 (en) 2007-09-04 2015-08-18 Compugen Ltd. Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics
US9428574B2 (en) 2011-06-30 2016-08-30 Compugen Ltd. Polypeptides and uses thereof for treatment of autoimmune disorders and infection
US9617336B2 (en) 2012-02-01 2017-04-11 Compugen Ltd C10RF32 antibodies, and uses thereof for treatment of cancer
US9714294B2 (en) 2010-05-27 2017-07-25 Genmab A/S Monoclonal antibodies against HER2 epitope
CN107922494A (en) * 2015-07-28 2018-04-17 钜川生物医药 Anti- 1 antibody of PD and its application
CN110305217A (en) * 2018-03-27 2019-10-08 广州爱思迈生物医药科技有限公司 Bispecific antibody and its application
EP3752536A4 (en) * 2018-02-13 2022-05-11 Agency for Science, Technology and Research Anti-her2 antibodies
US11578141B2 (en) 2011-04-20 2023-02-14 Genmab A/S Bispecific antibodies against HER2 and CD3
IL267022B2 (en) * 2010-05-27 2023-06-01 Genmab As Monoclonal antibodies aganist her2
US11738081B2 (en) 2015-03-31 2023-08-29 Medimmune Limited Polynucleotides encoding IL33 antibodies and methods of using the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018233624A1 (en) * 2017-06-20 2018-12-27 和迈生物科技有限公司 Anti-her2 nanobody and coding sequence and use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821337A (en) * 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821337A (en) * 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DISATNIK M-H ET AL: "DISTINCT RESPONSES OF PROTEIN KINASE C ISOZYMES TO C-ERBB-2 ACTIVATION IN SKBR-3 HUMAN BREAST CARCINOMA CELLS" CELL GROWTH AND DIFFERENTIATION, THE ASSOCIATION, PHILADELPHIA, PA, US, vol. 5, no. 8, 1 August 1994 (1994-08-01), pages 873-880, XP000961397 ISSN: 1044-9523 *
HARWERTH I-M ET AL: "MONOCLONAL ANTIBODIES AGAINST THE EXTRACELLULAR DOMAIN OF THE ERBB-2 RECEPTOR FUNCTION AS PARTIAL LIGAND AGONISTS" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,; US, vol. 267, no. 21, 1 January 1992 (1992-01-01), pages 15160-15167, XP009107550 ISSN: 0021-9258 *
LITTLE M ET AL: "Of mice and men: hybridoma and recombinant antibodies" IMMUNOLOGY TODAY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 21, no. 8, 1 August 2000 (2000-08-01), pages 364-370, XP004215163 ISSN: 0167-5699 *
MCKEAGE K ET AL: "Trastuzumab: a review of its use in the treatment of metastatic breast cancer overexpressing HER2" DRUGS, ADIS INTERNATIONAL LTD, vol. 62, no. 1, 1 January 2002 (2002-01-01), pages 209-243, XP009088461 ISSN: 0012-6667 *
MOLINA MA ET AL: "Trastuzumab (Herceptin), a Humanized Anti-HER2 Receptor Monoclonal Antibody, Inhibits Basal and Activated HER2 Ectodomain Cleavage in Breast Cancer Cells" CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD.; US, vol. 61, 1 January 2001 (2001-01-01), pages 4744-4749, XP002408598 ISSN: 0008-5472 *
PERRY C M ET AL: "TRASTUZUMAB" BIODRUGS, AUCKLAND, NZ, vol. 12, no. 2, 1 August 1999 (1999-08-01), pages 129-135, XP002948250 ISSN: 1173-8804 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9555087B2 (en) 2007-09-04 2017-01-31 Compugen Ltd Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics
US9107862B2 (en) 2007-09-04 2015-08-18 Compugen Ltd. Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics
US9375466B2 (en) 2007-09-04 2016-06-28 Compugen Ltd Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics
US10098934B2 (en) 2007-09-04 2018-10-16 Compugen Ltd Polypeptides and polynucleotides, and uses thereof as a drug target for producing drugs and biologics
CN107253992A (en) * 2010-05-27 2017-10-17 根马布股份公司 For HER2 monoclonal antibody
IL267022B2 (en) * 2010-05-27 2023-06-01 Genmab As Monoclonal antibodies aganist her2
US9714294B2 (en) 2010-05-27 2017-07-25 Genmab A/S Monoclonal antibodies against HER2 epitope
AU2016201676B2 (en) * 2010-05-27 2017-09-14 Genmab A/S Monoclonal antibodies against HER2
US11091553B2 (en) 2010-05-27 2021-08-17 Genmab A/S Monoclonal antibodies against HER2
US9862769B2 (en) 2010-05-27 2018-01-09 Genmab A/S Monoclonal antibodies against HER2
WO2011147986A1 (en) * 2010-05-27 2011-12-01 Genmab A/S Monoclonal antibodies against her2
EP3539988A3 (en) * 2010-05-27 2019-12-04 Genmab A/S Monoclonal antibodies against her2
CN107253992B (en) * 2010-05-27 2022-03-11 根马布股份公司 Monoclonal antibody against HER2
US11046771B2 (en) 2010-05-27 2021-06-29 Genmab A/S Monoclonal antibodies against HER2
US11578141B2 (en) 2011-04-20 2023-02-14 Genmab A/S Bispecific antibodies against HER2 and CD3
US9428574B2 (en) 2011-06-30 2016-08-30 Compugen Ltd. Polypeptides and uses thereof for treatment of autoimmune disorders and infection
US9617336B2 (en) 2012-02-01 2017-04-11 Compugen Ltd C10RF32 antibodies, and uses thereof for treatment of cancer
US11738081B2 (en) 2015-03-31 2023-08-29 Medimmune Limited Polynucleotides encoding IL33 antibodies and methods of using the same
CN107922494B (en) * 2015-07-28 2021-05-07 上海昀怡健康科技发展有限公司 anti-PD-1 antibodies and uses thereof
CN107922494A (en) * 2015-07-28 2018-04-17 钜川生物医药 Anti- 1 antibody of PD and its application
US11945877B2 (en) 2018-02-13 2024-04-02 Agency For Science, Technology And Research Anti-HER2 antibodies
EP3752536A4 (en) * 2018-02-13 2022-05-11 Agency for Science, Technology and Research Anti-her2 antibodies
CN110305217A (en) * 2018-03-27 2019-10-08 广州爱思迈生物医药科技有限公司 Bispecific antibody and its application
US11718671B2 (en) 2018-03-27 2023-08-08 Excelmab, Inc. Bispecific antibody and uses thereof
CN110305217B (en) * 2018-03-27 2022-03-29 广州爱思迈生物医药科技有限公司 Bispecific antibodies and uses thereof

Also Published As

Publication number Publication date
WO2009055074A3 (en) 2009-09-11
WO2009055074A8 (en) 2009-08-06

Similar Documents

Publication Publication Date Title
US20090258005A1 (en) Therapeutic compositions and methods
JP7301106B2 (en) Monoclonal antibody against fibroblast growth factor receptor 2
WO2009055074A2 (en) Erbb2 binding proteins and use thereof
US20090304590A1 (en) Therapeutic compositions and methods
CA2678181C (en) Antibodies against erbb3 and uses thereof
US8043618B2 (en) Fibroblast growth factor receptor-3 (FGFR-3) inhibitors and methods of treatment
US20110150759A1 (en) Monoclonal antibody 175 tageting the egf receptor and derivatives and uses thereof
EA036739B1 (en) Antibodies for epidermal growth factor receptor 3 (her3)
NZ575674A (en) Anti-VEGF antibodies
CA2705923A1 (en) Antibodies to lrp6
JP2014504850A (en) Production, characterization and use of anti-HER3 antibodies
RU2653443C2 (en) Bispecific anti-her2 / anti-her3 antibodies
AU2012201667B2 (en) Anti-VEGF antibodies
TW202003038A (en) Bispecific antibody that specifically binds to subdomains IV and II of extracellular domain of human HER2
AU2008287335B8 (en) Monoclonal antibody 175 targeting the EGF receptor and derivatives and uses thereof
AU2014259593A1 (en) Anti-vegf antibodies
AU2012204022A1 (en) Binding polypeptides and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08842686

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08842686

Country of ref document: EP

Kind code of ref document: A2