ISOLATED MONOCLONAL ANTIBODY OR ANTIGEN BINDING FRAGMENT OF THE SAME TO THE HER3 RECEPTOR, ITS USE A

专利摘要:
antibodies to the epidermal growth factor 3 (her3) receptor, their use and fragments, as well as pharmaceutical compositions that comprise them. the present invention relates to antibodies or fragments thereof that target a conformational epitope of a her receptor. in particular, the present invention relates to antibodies or fragments thereof that target a conformational epitope of the her3 receptor and the compositions and methods of using them.
公开号:BR112013004012B1
申请号:R112013004012-2
申请日:2011-08-22
公开日:2021-03-23
发明作者:Winfried ELIS；Seth Ettenberg；Andrew Paul Garner；Nicole HAUBST；Christian Carsten Silvester Kunz；Elizabeth Anne Reisinger Sprague
申请人:Novartis Ag；
IPC主号:

专利说明:

[0001] [001] This patent application claims priority to provisional Patent Application 61/375, 408, filed on August 20, 2010, the content of which is included in its entirety. Field of the present invention
[0002] [002] The present invention relates, in general, to antibodies or fragments thereof that interact with the family of receptors of, for example, the HER3 receptor. In particular, it refers to antibodies or fragments thereof that recognize a conformational epitope of the HER receptor (for example, HER3) comprising residues from both domains 2 and 4, resulting in inhibition of both ligand-dependent and signal transduction regardless of the ligand. The present invention also relates to antibodies and fragments thereof that bind to HER receptors (for example, HER3 receptor) simultaneously with a ligand (for example, neuregulin), while preventing activation induced by the signal transduction ligand . Background of the present invention
[0003] [003] Human epidermal growth factor receptor 3 (ErbB3, also known as HER3) is a protein tyrosine kinase receptor and belongs to the subfamily receptor epidermal growth factor (EGFR) of protein tyrosine kinase receptors , which also includes EGFR (HER1, ErbB1), HER2 (ErbB2, Neu) and HER4 (ErbB4) (Plowman et al, (1990) Proc Natl Acad Sci US 87: 4905 to 4909, Kraus et al, Proc (1989) Natl Acad Sci. US 86: 9193 to 9197, and Kraus et al, Proc (1993) Natl Acad Sci US 90: 2900 to 2904). Like the prototype epidermal growth factor receptor, the HER3 transmembrane receptor consists of an extracellular ligand (ECD) binding domain, a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain. Unlike the other members of its family, the HER3 kinase domain exhibits very low intrinsic kinase activity.
[0004] [004] The neuregulin 1 (NRG) or neuregulin2 ligands bind to the extracellular domain of HER3 and activate the receptor-mediated signaling pathway by promoting dimerization with dimerization partners, such as HER2. Heterodimerization results in the activation and transfosphorylation of the intracellular HER3 domain and is a means not only for signal diversification, but also with regard to signal amplification. In addition, HER3 heterodimerization can also occur in the absence of activation ligands and this is usually called independent of the HER3 activation ligand. For example, when HER2 is expressed at high levels as a result of gene amplification (eg, breast, ovary, lung or gastric cancer) spontaneous HER2 / HER3 dimers can be formed. In this situation, HER2 / HER3 is considered the most active ErbB signaling dimer and is therefore highly transformative.
[0005] [005] Increased HER3 has been found in several types of cancer, such as breast, lung, gastrointestinal and pancreas. It is interesting to note that a correlation between the expression of HER2 / HER3 and the progression of a non-invasive form to an invasive phase has been shown (Alimandi et al, (1995) Oncogene 10: 1813 to 1821; Cancer 87: 487 to 498; Naidu et al, (1988) Br. J. Cancer 78: 1385 to 1390). Therefore, agents that interfere with HER3-mediated signaling are needed. Summary of the present invention
[0006] [006] The present invention is based on the discovery of antigen-binding proteins (for example, antibodies or fragments thereof) that bind to a conformational epitope of the HER3 receptor comprising amino acid residues within domain 2 and domain 4 of HER3. This binding of antibodies or their fragments to domain 2 and domain 4 stabilizes the HER3 receptor in an inactive or closed conformation such that HER3 activation is inhibited. Surprisingly, the binding of antibodies or their fragments to this conformational epitope blocks both ligand-dependent (e.g., neuregulin) and the HER3 ligand-independent signaling pathways. In addition, inhibitor-mediated inhibition of ligand-induced signaling occurs without blocking ligand binding (that is, both the ligand and antibody can bind HER3), presumably because HER3 cannot undergo conformational rearrangements necessary for activation.
[0007] [007] Thus, in one aspect, the present invention relates to an isolated antibody or a fragment thereof that binds to an inactive state of an HER receptor, in which the antibody or a fragment thereof both blocks the transduction of ligand-dependent and ligand-independent signal. In one embodiment, the antibody, or a fragment thereof, stabilizes the HER receptor in an inactive state.
[0008] [008] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of an HER receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the HER receptor, and where the antibody or a fragment thereof blocks both ligand-dependent and ligand-independent signal transductions. In one embodiment, the antibody or a fragment thereof binds to the inactive state of the HER receptor. In one embodiment, the antibody or a fragment of it binds to the active state of the HER receptor and triggers it in the inactive state. In another embodiment, the antibody or a fragment thereof stabilizes the HER receptor in an inactive state. The HER receptor is selected from the group consisting of HER1, HER2, HER3 and HER4. The antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody and a synthetic antibody.
[0009] [009] Another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of a HER receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the HER receptor, where antibody binding stabilizes the HER receptor in an inactive state, and where an HER ligand can simultaneously bind to a ligand-binding site on the HER receptor. In one embodiment, binding to the HER ligand to the ligand binding site does not induce a conformational change in the HER receptor to an active state. In another embodiment, binding to the HER ligand to the ligand binding site does not activate signal transduction.
[0010] [0010] In one embodiment, the ligand is HER selected from the group consisting of neuregulin 1 (NRG), neuregulin 2, neuregulin 3, neuregulin 4, betacellulin, heparin-binding epidermal growth factor, epiregulin, growth factor epidermal, amphiregulin, and transforming growth factor alpha.
[0011] [0011] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of a HER receptor, wherein the conformational epitope comprises the amino acid residues within domain 2 and domain 4 of the HER receptor, in which binding to the antibody stabilizes the HER receptor in an inactive state such that the HER receptor fails to dimerize with a co-receptor in order to form a receptor-receptor complex. The inability to form a receptor-receptor complex prevents the activation of signal transduction, both dependent on the ligand and independent of the ligand.
[0012] [0012] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of an HER receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the HER receptor, in which the binding of the antibody to the HER receptor allows dimerization with a co-receptor in order to form an inactive receptor-receptor complex. The formation of the receptor-receptor complex prevents the activation of signal transduction independent of the ligand.
[0013] [0013] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that binds to an inactive conformation of an HER3 receptor, wherein the antibody blocks both ligand-dependent and ligand-independent signal transductions . In one embodiment, the antibody, or a fragment thereof, stabilizes the HER3 receptor in an inactive state.
[0014] [0014] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of an HER3 receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the HER3 receptor, and where the antibody or a fragment thereof blocks both ligand-dependent and ligand-independent signal transductions. In one embodiment, the antibody or a fragment thereof binds to the inactive state of the HER3 receptor. In another embodiment, the antibody or a fragment thereof stabilizes the HER3 receptor, in an inactive state. The antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody and a synthetic antibody.
[0015] [0015] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of an HER3 receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 HER3 receptor, in which the binding of the antibody stabilizes the HER3 receptor in an inactive state, and in which an HER3 ligand can simultaneously bind to a ligand binding site on the HER3 receptor. In one embodiment, the binding of the HER3 ligand to the ligand binding site does not induce a conformational change in the HER3 receptor to an active state. In another embodiment, the ligation of the HER3 ligand to the ligand ligation site does not activate signal transduction. In one embodiment, the HER3 ligand is selected from the group consisting of neuregulin 1 (NRG), neuregulin 2, betacellulin, heparin-binding epidermal growth factor and epiregulin.
[0016] [0016] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of an HER3 receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the HER3 receptor, and where the antibody or a fragment thereof blocks both ligand-dependent and ligand-independent signal transductions. In one embodiment, the antibody or a fragment thereof binds to the inactive state of the HER3 receptor. In another embodiment, the antibody or a fragment thereof stabilizes the HER3 receptor in an inactive state.
[0017] [0017] In another aspect, the present invention relates to an isolated antibody or the fragment thereof that binds to a conformational epitope of the HER3 receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the HER3 receptor, in which domain 2, comprises a dimerization circuit, and in which the antibody or fragment blocks both ligand-dependent and ligand-independent signal transductions. In one embodiment, the antibody, or a fragment thereof, stabilizes the HER3 receptor in an inactive state. In one embodiment, the conformational epitope comprises amino acid residues 265 to 277, 315 (from domain 2), 571,582-584, 596-597, 600 to 602, 609-615 (domain 4) or a subset of these. In one embodiment, the VH of the antibody or a fragment thereof binds to at least one of the following residues: HER3 Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276, Asn275, His277, Asn315, Asp571, Pro583 , His584, Ala596, Lys597. In one embodiment, the VL of the antibody or a fragment thereof binds to at least one of the following residues: HER3 Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Lys602, Ile600, Arg611, Glu609, Pro612, Cys613 , His614, Glu615.
[0018] [0018] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of the first HER receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the first HER receptor, where binding of the antibody or a fragment thereof to the first HER receptor in the absence of a HER receptor ligand reduces the ligand-independent formation of a first HER receptor protein complex - second cell HER receptor which expresses the first HER receptor and the second HER receptor. In one embodiment, the antibody, or a fragment thereof, stabilizes the first HER receptor in an inactive state such that the first HER receptor fails in order to form dimers with the second HER receptor in order to form a protpein complex. of the first HER receiver - second HER receiver. In one embodiment, failure to form a protein complex of the first HER receptor - second HER receptor prevents activation of signal transduction. In one embodiment, the first HER is selected from the group consisting of HER1, HER2, HER3, and HER4. In one embodiment, the second HER is selected from the group consisting of HER1, HER2, HER3, and HER4.
[0019] [0019] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of the HER3 receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of HER3 , in which the binding of the antibody or its fragment to the HER3 receptor in the absence of an HER3 ligand independently reduces the formation of the ligand of a HER2 - HER3 protein complex in a cell that expresses HER2 and HER3. In one embodiment, the antibody, or a fragment thereof, stabilizes the HER3 receptor in an inactive state such that the HER3 receptor fails to dimerize with the HER2 receptor, in order to form a HER2 - HER3 protein complex. In one embodiment, failure to form a HER2 - HER3 protein complex prevents activation of signal transduction. In one embodiment, the antibody, or a fragment thereof, stabilizes the HER3 receptor in an inactive state such that the HER3 receptor can still dimerize with HER2, but forms an inactive HER2 - HER3 protein complex. In one embodiment, the formation of an inactive HER2 -HER3 protein complex prevents activation of signal transduction.
[0020] [0020] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of the first HER receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of the first receptor, in which binding the antibody or a fragment thereof to the first HER receptor in the presence of a HER ligand reduces the ligand-dependent formation of a protein complex of the first HER receptor - the second HER receptor of a cell that expresses the first HER receiver and second HER receiver. In one embodiment, the antibody, or a fragment thereof, stabilizes the first HER receptor in an inactive state such that the HER receptor fails to form dimers with the second HER receptor in the presence of a ligand for the purpose of forming a protein complex of the first HER receptor - second HER receptor. In one embodiment, failure to form a protein complex of the first HER receptor - second HER receptor prevents activation of signal transduction. In one embodiment, the HER ligand is selected from the group consisting of neuregulin 1 (NRG), neuregulin 2, neuregulin 3, neuregulin 4, betacellulin, heparin-linked epidermal growth factor, epiregulin, epidermal growth factor, amphiregulin , and transforming growth factor alpha. In one embodiment, the first HER is selected from the group consisting of HER1, HER2, HER3, and HER4. In one embodiment, the second HER is selected from the group consisting of HER1, HER2, HER3, and HER4.
[0021] [0021] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of the HER3 receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of HER3 , in which the binding of the antibody or its fragment to the HER3 receptor in the presence of an HER3 ligand reduces the ligand-dependent formation of a HER2 - HER3 protein complex in a cell that expresses HER2 and HER3. The ligand is selected from the group consisting of neuregulin 1 (NRG), and neuregulin 2. In one embodiment, the antibody or a fragment thereof, stabilizes the HER3 receptor in an inactive state such that the HER3 receptor fails to dimerize with the HER2 receptor in the presence of an HER3 ligand, in order to form a HER2 - HER3 protein complex. In one embodiment, failure to form a HER2 - HER3 protein complex prevents activation of signal transduction.
[0022] [0022] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of the HER3 Receptor, wherein the conformational epitope comprises amino acid residues within domain 2 and domain 4 of HER3 , and wherein the antibody or fragment thereof inhibits HER3 phosphorylation, as assessed by the ligand-independent HER3 phosphorylation assay. In one embodiment, the HER3 phosphorylation assay independent of the HER2 ligand uses the amplified cells, where the amplified HER2 cells are the SK-BR-3 cells.
[0023] [0023] In another aspect, the present invention relates to an isolated antibody or a fragment thereof that recognizes a conformational epitope of the HER3 Receptor, wherein the conformational epitope comprises the amino acid residues within domain 2 and domain 4 of HER3 , and wherein the antibody or fragment thereof inhibits HER3 phosphorylation, as assessed by the HER3 ligand-dependent phosphorylation assay. In one embodiment, the HER3 ligand-dependent phosphorylation assay uses MCF7 cells stimulated with neuregulin (NRG).
[0024] [0024] In another aspect, the present invention relates to the isolated antibody or a fragment thereof to an HER3 receptor, which has a dissociation (KD) of at least 1 x 107 M-1, 108 M-1, 109 M -1, 1010 M -1, 1011 M -1, 1012 M -1, 1013 M -1. In one embodiment, the antibody or fragment thereof inhibits HER3 phosphorylation as measured by in vitro human HER3 binding tests in a phosphorylation assay selected from the group consisting of phospho-HER3 and phospho-Akt.
[0025] [0025] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor, where the crosses compete with an antibody described in table 1; an antibody or fragment thereof that interacts with (for example, by means of impairment, steric binding, stabilization / destabilization, spatial distribution) the same epitope, as an antibody described in Table 1. In one embodiment, the antibody or a fragment thereof is a monoclonal antibody. In another embodiment, the antibody or a fragment thereof is a human or humanized antibody. In another embodiment, the antibody or a fragment thereof is a chimeric antibody. In one embodiment, the antibody or a fragment thereof comprises a human heavy chain constant region and a human constant light chain region. In one embodiment, the antibody or a fragment thereof is a single chain antibody. In another embodiment, the antibody or a fragment thereof is a Fab fragment. In yet another embodiment, the antibody or a fragment thereof is an scFv. In one embodiment, the antibody or a fragment of it binds to both human HER3 and cinomologist HER3. In one embodiment, the antibody or a fragment thereof is an IgG isotype. In another embodiment, the antibody or a fragment thereof comprises a structure in which amino acids have been replaced in the antibody structure from the respective human VH or germline VL sequences.
[0026] [0026] In one aspect, the present invention relates to an isolated antibody or a fragment thereof comprising HER3 1, 2, 3, 4, 5, or 6 CDRs calculated by means of Kabat Chothia or by means of any of the antibodies in the Table 1.
[0027] [0027] In another aspect, the present invention relates to an isolated antibody or fragment thereof to the HER3 Receptor comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 58, SEQ ID NO: 64, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 94, SEQ ID NO: 100, SEQ ID NO: 112, SEQ ID NO: 118, SEQ ID NO: 130, SEQ ID NO: 136, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 220, SEQ ID NO: 238, SEQ ID NO: 256, SEQ ID NO: 274, SEQ ID NO: 292, SEQ ID NO: 310, SEQ ID NO: 328, SEQ ID NO: 346, and SEQ ID NO: 364.
[0028] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 15 and a VL comprising SEQ ID NO: 14, or a amino acid sequence with the identity of 97 to 99% of the same.
[0029] [0029] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 33 and a VL comprising SEQ ID NO: 32, or a amino acid sequence with the identity of 97 to 99% of the same.
[0030] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 51 and a VL comprising SEQ ID NO: 50, or a amino acid sequence with the identity of 97 to 99% of the same.
[0031] [0031] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 69 and a VL comprising SEQ ID NO: 68, or a amino acid sequence with the identity of 97 to 99% of the same.
[0032] [0032] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 87 and a VL comprising SEQ ID NO: 86, or a amino acid sequence with the identity of 97 to 99% of the same.
[0033] [0033] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 105 and a VL comprising SEQ ID NO: 104, or a amino acid sequence with the identity of 97 to 99% of the same.
[0034] [0034] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 123 and a VL comprising SEQ ID NO: 122, or a amino acid sequence with the identity of 97 to 99% of the same.
[0035] [0035] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 141 and a VL comprising SEQ ID NO: 140, or a amino acid sequence with the identity of 97 to 99% of the same.
[0036] [0036] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 159 and a VL comprising SEQ ID NO: 158, or a amino acid sequence with the identity of 97 to 99% of the same.
[0037] [0037] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 177 and a VL comprising SEQ ID NO: 176, or a amino acid sequence with the identity of 97 to 99% of the same.
[0038] [0038] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 195 and a VL comprising SEQ ID NO: 194, or a amino acid sequence with the identity of 97 to 99% of the same.
[0039] [0039] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 213 and a VL comprising SEQ ID NO: 212, or a amino acid sequence with the identity of 97 to 99% of the same.
[0040] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 231 and a VL comprising SEQ ID NO: 230, or a amino acid sequence with the identity of 97 to 99% of the same.
[0041] [0041] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 249, and a VL comprising SEQ ID NO: 248, or a sequence of amino acids with the identity of 97 to 99% of the same.
[0042] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 267, and a VL comprising SEQ ID NO: 266, or a sequence of amino acids with the identity of 97 to 99% of the same.
[0043] [0043] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 285 and a VL comprising SEQ ID NO: 284, or a amino acid sequence with the identity of 97 to 99% of the same.
[0044] [0044] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 303 and a VL comprising SEQ ID NO: 302, or a amino acid sequence with the identity of 97 to 99% of the same.
[0045] [0045] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 321 and a VL comprising SEQ ID NO: 320, or a amino acid sequence with the identity of 97 to 99% of the same.
[0046] [0046] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 339 and a VL comprising SEQ ID NO: 338, or a amino acid sequence with the identity of 97 to 99% of the same.
[0047] [0047] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 357 and a VL comprising SEQ ID NO: 356, or a amino acid sequence with the identity of 97 to 99% of the same.
[0048] [0048] In another aspect, the present invention relates to an isolated antibody or a fragment thereof of the HER3 receptor that the antibody comprises a VH comprising SEQ ID NO: 375 and a VL comprising SEQ ID NO: 374, or a amino acid sequence with the identity of 97 to 99% of the same.
[0049] [0049] In another aspect, the present invention relates to an isolated antibody or the fragment thereof comprising a variable heavy chain sequence having SEQ ID NO: 493.
[0050] In another aspect, the present invention relates to an isolated antibody or the fragment thereof comprising a light chain variable sequence having SEQ ID NO: 494.
[0051] [0051] In another aspect, the present invention relates to an isolated antibody or fragment thereof comprising a heavy chain variable sequence that has SEQ ID NO: 493 and a light chain variable sequence that has SEQ ID NO. : 494.
[0052] [0052] In another aspect, the present invention relates to an isolated antibody or a fragment thereof for HER3 Receptor with a heavy chain variable region variant comprising CDR1, CDR2 and CDR3, wherein the variant has at least 1 to 4 amino acid changes in a CDR1, CDR2, or CDR3.
[0053] [0053] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 2; CDR2 of SEQ ID NO: 3; CDR3 of SEQ ID NO: 4, a variable region of the CDR1 light chain of SEQ ID NO: 5; CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 7.
[0054] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 20; CDR2 of SEQ ID NO: 21; CDR3 of SEQ ID NO: 22, a variable region of the light chain CDR1 of SEQ ID NO: 23, CDR2 of SEQ ID NO: 24, and CDR3 of SEQ ID NO: 25.
[0055] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 38; CDR2 of SEQ ID NO: 39; CDR3 of SEQ ID NO: 40, a variable region of the CDR1 light chain of SEQ ID NO: 41; CDR2 of SEQ ID NO: 42, and CDR3 of SEQ ID NO: 43.
[0056] [0056] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 56; CDR2 of SEQ ID NO: 57; CDR3 of SEQ ID NO: 58, a variable region of the light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO: 60, and CDR3 of SEQ ID NO: 61.
[0057] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 74, CDR2 of SEQ ID NO: 75; CDR3 of SEQ ID NO: 76, a variable region of the CDR1 light chain of SEQ ID NO: 77; CDR2 of SEQ ID NO: 78, and CDR3 of SEQ ID NO: 79.
[0058] [0058] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 92; CDR2 of SEQ ID NO: 93; CDR3 of SEQ ID NO: 94, a variable region of the CDR1 light chain of SEQ ID NO: 95; CDR2 of SEQ ID NO: 96, and CDR3 of SEQ ID NO: 97.
[0059] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 110, CDR2 of SEQ ID NO: 111; CDR3 of SEQ ID NO: 112, a variable region of the CDR1 light chain of SEQ ID NO: 113, CDR2 of SEQ ID NO: 114, and CDR3 of SEQ ID NO: 115.
[0060] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 128, CDR2 of SEQ ID NO: 129; CDR3 of SEQ ID NO: 130, a variable region of the CDR1 light chain of SEQ ID NO: 131, CDR2 of SEQ ID NO: 132, and CDR3 of SEQ ID NO: 133.
[0061] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 146, CDR2 of SEQ ID NO: 147; CDR3 of SEQ ID NO: 148, a variable region of the CDR1 light chain of SEQ ID NO: 149, CDR2 of SEQ ID NO: 150, and CDR3 of SEQ ID NO: 151.
[0062] [0062] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 164, CDR2 of SEQ ID NO: 165; CDR3 of SEQ ID NO: 166, a variable region of the CDR1 light chain of SEQ ID NO: 167, CDR2 of SEQ ID NO: 168, and CDR3 of SEQ ID NO: 169.
[0063] [0063] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 182, CDR2 of SEQ ID NO: 183; CDR3 of SEQ ID NO: 184, a variable region of the CDR1 light chain of SEQ ID NO: 185, CDR2 of SEQ ID NO: 186, and CDR3 of SEQ ID NO: 187.
[0064] [0064] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 200, CDR2 of SEQ ID NO: 201; CDR3 of SEQ ID NO: 202, a variable region of the light chain CDR1 of SEQ ID NO: 203, CDR2 of SEQ ID NO: 204, and CDR3 of SEQ ID NO: 205.
[0065] [0065] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 218, CDR2 of SEQ ID NO: 219; CDR3 of SEQ ID NO: 220, a variable region of the CDR1 light chain of SEQ ID NO: 221, CDR2 of SEQ ID NO: 222, and CDR3 of SEQ ID NO: 223.
[0066] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 236, CDR2 of SEQ ID NO: 237; CDR3 of SEQ ID NO: 238, a variable region of the CDR1 light chain of SEQ ID NO: 239, CDR2 of SEQ ID NO: 240, and CDR3 of SEQ ID NO: 241.
[0067] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 254, CDR2 of SEQ ID NO: 255; CDR3 of SEQ ID NO: 256, a variable region of the CDR1 light chain of SEQ ID NO: 257, CDR2 of SEQ ID NO: 258, and CDR3 of SEQ ID NO: 259.
[0068] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 272, CDR2 of SEQ ID NO: 273; CDR3 of SEQ ID NO: 274, a variable region of the CDR1 light chain of SEQ ID NO: 275, CDR2 of SEQ ID NO: 276, and CDR3 of SEQ ID NO: 277.
[0069] [0069] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 290, CDR2 of SEQ ID NO: 291; CDR3 of SEQ ID NO: 292, a variable region of the CDR1 light chain of SEQ ID NO: 293, CDR2 of SEQ ID NO: 294, and CDR3 of SEQ ID NO: 295.
[0070] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 308, CDR2 of SEQ ID NO: 309; CDR3 of SEQ ID NO: 310, a variable region of the CDR1 light chain of SEQ ID NO: 311, CDR2 of SEQ ID NO: 312, and CDR3 of SEQ ID NO: 313.
[0071] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 326, CDR2 of SEQ ID NO: 327; CDR3 of SEQ ID NO: 328, a variable region of the CDR1 light chain of SEQ ID NO: 329, CDR2 of SEQ ID NO: 330, and CDR3 of SEQ ID NO: 331.
[0072] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 344, CDR2 of SEQ ID NO: 345; CDR3 of SEQ ID NO: 346, a variable region of the CDR1 light chain of SEQ ID NO: 347, CDR2 of SEQ ID NO: 348, and CDR3 of SEQ ID NO: 349.
[0073] In another aspect, the present invention relates to an isolated antibody or a fragment thereof, which comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 362, CDR2 of SEQ ID NO: 363; CDR3 of SEQ ID NO: 364, a variable region of the CDR1 light chain of SEQ ID NO: 365, CDR2 of SEQ ID NO: 366, and CDR3 of SEQ ID NO: 367.
[0074] [0074] In one embodiment, an antibody fragment that binds to HER3 is selected from the group consisting of Fab, F (ab2) ', F (ab) 2', scFv, VHH, VH, VL, DABS.
[0075] [0075] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody or fragment and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antibody, a small molecule, an inhibitor or an inhibitor of mTOR PI3Kinase. In one embodiment, the pharmaceutical composition comprises the antibody or fragment thereof of the present invention and an HER1 inhibitor, including, but not limited to, Matuzumab (EMD72000), Erbitux ® / Cetuximab, Vectibix ® / Panitumumab, mAb 806, Nimotuzumab, Iressa® / Gefitinib, CI-1033 (PD183805), lapatinib (GW-572016), Tykerb® / lapatinib ditosylate, Tarceva® / erlotinib HCL (OSI-774), PKI-166, and Tovok®.
[0076] [0076] In one embodiment, the pharmaceutical composition comprises the antibody or a fragment of the present invention and an HER2 inhibitor, including, but not limited to, pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib or ditosylate / lapatinib Tykerb ® .
[0077] [0077] In one embodiment, the pharmaceutical composition comprises the antibody or fragment thereof of the present invention and an HER3 inhibitor, including, but not limited to, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG ), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech); small molecules that inhibit HER3.
[0078] [0078] In one embodiment, the pharmaceutical composition comprises the antibody or fragment of the present invention and an HER4 inhibitor.
[0079] [0079] In one embodiment, the pharmaceutical composition comprises the antibody or fragment thereof of the present invention and a PI3 kinase inhibitor, including, but not limited to, GDC 0941 BEZ235, BMK120 and BYL719.
[0080] [0080] In one embodiment, the pharmaceutical composition comprises the antibody or fragment of the present invention and an mTOR inhibitor, including, but not limited to, Temsirolimus / Torisel ®, ridaforolimus / Deforolimus, AP23573, MK8669, everolimus / Affinitor ®. In another aspect, the present invention relates to a method of treating a cancer which comprises selecting a subject who has a cancer that expresses HER3, administering to the person an effective amount of a composition comprising an antibody or a fragment thereof, selected from any of the preceding claims. In one embodiment, the subject is a human being.
[0081] [0081] In another aspect, the present invention relates to a method of treating a cancer which comprises selecting a subject who has a cancer that expresses HER3, administering to the person an effective amount of a composition comprising an antibody or a fragment of the same, selected from any of the preceding claims, characterized in that the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear cell sarcoma soft tissue, malignant mesothelioma, neurofibromatosis, kidney cancer, melanoma. In one embodiment, cancer is breast cancer.
[0082] [0082] In another aspect, the present invention relates to a method of treating a cancer which comprises selecting a subject who has a cancer that expresses HER3, administering to said individual an effective amount of a composition comprising a combination of antibodies or the fragments thereof described in Table 1 that bind to HER3.
[0083] [0083] In another aspect, the present invention relates to a method of treating a cancer which comprises selecting a subject who has a cancer that expresses HER3, administering to said individual an effective amount of a composition comprising an antibody or a fragment of it that binds to HER3 and inhibits signal transduction dependent on the HER3 ligand and signal transduction independent of the ligand.
[0084] [0084] In another aspect, the present invention relates to the use of an antibody or a fragment thereof of any of the preceding claims in the manufacture of a medicament for the treatment of cancer mediated by means of a ligand-dependent signal transduction HER3 or signal transduction pathway independent of the ligand selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, soft-tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis , kidney cancer and melanoma.
[0085] [0085] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 15 and VL of SEQ ID NO: 14 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0086] [0086] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32 for use in the treatment of cancer mediated by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0087] [0087] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 51 and VL of SEQ ID NO: 50, for use in the treatment of a mediated cancer by means of a ligand-dependent signal transduction HER3 or via ligand-independent signal transduction.
[0088] [0088] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 69 and VL of SEQ ID NO: 68, for use in the treatment of a mediated cancer by means of a ligand-dependent signal transduction HER3 or via ligand-independent signal transduction.
[0089] [0089] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 87 and VL of SEQ ID NO: 86 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0090] [0090] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 105 and VL of SEQ ID NO: 104, for use in the treatment of a mediated cancer by means of a ligand-dependent signal transduction HER3 or via ligand-independent signal transduction.
[0091] [0091] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 123 and VL of SEQ ID NO: 122 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0092] [0092] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 141 and VL of SEQ ID NO: 140, for use in the treatment of a mediated cancer by means of a ligand-dependent signal transduction HER3 or via ligand-independent signal transduction.
[0093] [0093] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 151 and VL of SEQ ID NO: 158 for use in the treatment of cancer mediated by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0094] [0094] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 177 and VL of SEQ ID NO: 176 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0095] [0095] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 195 and VL of SEQ ID NO: 194 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0096] [0096] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 213 and VL of SEQ ID NO: 212 for use in the treatment of a mediated cancer using a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0097] [0097] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 231 and VL of SEQ ID NO: 230 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0098] [0098] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 249 and VL of SEQ ID NO: 248 for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0099] [0099] In another aspect, the present invention relates to an antibody having SEQ ID NO: 267 VH and SEQ ID NO: 266 VL for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0100] [00100] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 285 and VL of SEQ ID NO: 284 for use in the treatment of a mediated cancer by means of a signal transduction dependent on the HER3 ligand or by means of a ligand-independent signal transduction.
[0101] [00101] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 303 and VL of SEQ ID NO: 302 for use in the treatment of a mediated cancer by means of a signal transduction dependent on the HER3 ligand or by means of a ligand-independent signal transduction.
[0102] [00102] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 321 and VL of SEQ ID NO: 320, for use in the treatment of a mediated cancer by means of a ligand-dependent signal transduction HER3 or via ligand-independent signal transduction.
[0103] [00103] In another aspect, the present invention relates to an antibody having SEQ ID NO: 339 VH and SEQ ID NO: 338 VL for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction.
[0104] [00104] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 357 and VL of SEQ ID NO: 356 for use in the treatment of a mediated cancer by means of a signal transduction dependent on the HER3 ligand or by means of a ligand-independent signal transduction.
[0105] [00105] In another aspect, the present invention relates to an antibody having SEQ ID NO: 375 VH and SEQ ID NO: 374 VL for use in the treatment of a mediated cancer by means of a HER3 ligand-dependent signal transduction or by means of a ligand-independent signal transduction. In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 15 and VL of SEQ ID NO: 14 for use as a medicament.
[0106] [00106] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32, for use as a medicament.
[0107] [00107] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 51 and VL of SEQ ID NO: 50, for use as a medicament.
[0108] [00108] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 69 and VL of SEQ ID NO: 68, for use as a medicament.
[0109] [00109] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 87 and VL of SEQ ID NO: 86, for use as a medicament.
[0110] [00110] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 105 and VL of SEQ ID NO: 104, for use as a medicament.
[0111] [00111] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 123 and VL of SEQ ID NO: 122 for use as a medicament.
[0112] [00112] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 141 and VL of SEQ ID NO: 140, for use as a medicament.
[0113] [00113] In another aspect, the present invention relates to an antibody having SEQ ID NO: 159 VH and SEQ ID NO: 158 VL for use as a medicament.
[0114] [00114] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 177 and VL of SEQ ID NO: 176 for use as a medicament.
[0115] [00115] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 195 and VL of SEQ ID NO: 194 for use as a medicament.
[0116] [00116] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 213 and VL of SEQ ID NO: 212 for use as a medicament.
[0117] [00117] In another aspect, the present invention relates to an antibody having SEQ ID NO: 231 VH and SEQ ID NO: 230 VL for use as a medicament.
[0118] [00118] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 249 and VL of SEQ ID NO: 248 for use as a medicament.
[0119] [00119] In another aspect, the present invention relates to an antibody having SEQ ID NO: 267 VH and SEQ ID NO: 266 VL for use as a medicament.
[0120] [00120] In another aspect, the present invention relates to an antibody having SEQ ID NO: 285 VH and SEQ ID NO: 284 VL for use as a medicament.
[0121] [00121] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 302 for use as a medicament: 303 and VL of SEQ ID NO.
[0122] [00122] In another aspect, the present invention relates to an antibody having VH of SEQ ID NO: 321 and VL of SEQ ID NO: 320, for use as a medicament.
[0123] [00123] In another aspect, the present invention relates to an antibody having SEQ ID NO: 339 VH and SEQ ID NO: 338 VL for use as a medicament.
[0124] [00124] In another aspect, the present invention relates to an antibody having SEQ ID NO: 357 VH and SEQ ID NO: 356 VL for use as a medicament.
[0125] [00125] In another aspect, the present invention relates to an antibody having SEQ ID NO: 375 VH and SEQ ID NO: 374 VL for use as a medicament. Brief Description of the Figures
[0126] [00126] Figure 1: Curves of the MOR10701 representative set obtained with human, rat, mouse and cino HER3
[0127] [00127] Figure 2: Determination of SK-BR-3 cell linkage by means of FACS titration
[0128] [00128] Figure 3: HER3 ELISA binding domain
[0129] [00129] Figure 4: Mapping of deuterium hydrogen exchange epitopes. A) HER3 peptides recovered in the ECD HDX-MS analysis sequence are indicated by the dashed lines. Potential N-linked glycosylation sites are highlighted. B) The relative degree of deuteration observed in the peptides identified through MS. C) The protected residues mapped on the published HER3 crystal structure.
[0130] [00130] Figure 5: A) HER3 / MOR09823 surface representation and HER3 / MOR09825 x-ray crystal structures. HER3 (in light gray) is in the closed conformation, and MOR09823 or MOR09825 (in darker gray) bind to both domains 2 and 4. B). Surface view of HER3 of structure HER3 / MOR09823 shown with a similar orientation as (A). MOR09823 has been omitted for clarity. C) structure HER3 / MOR09823 illustrated as a tape structure, seen in a 90 ° rotation from panels (A), (B) and (D). D) A ribbon representation of the inactive HER3 conformation recognized by MOR09823 Fab with a close-up of the domain 2 / domain 4 interface, highlighting the HER3 residues that are within 5A of FAB. E) determination of the binding of mutant HER3 / MOR10703 by means of ELISA titration.
[0131] [00131] Figure 6: Inhibition of the induced ligand (A) or HER3 phosphorylation (B) independent of the ligand.
[0132] [00132] Figure 7: Inhibition of HER3 dependent downstream of signaling pathways in amplified HER2 cell lines.
[0133] [00133] Figure 8: The impact of HER3 on the inhibition of cell growth in A) BT-474 and B) neuregulin stimulated from MCF-7 cells.
[0134] [00134] Figure 9: The neuregulin effect of MOR09823 and MOR09825 on binding to MCF7 cells.
[0135] [00135] Figure 10: Impact of MOR09823 binding to the formation of the HER3 / neuregulin complex evaluated by BiacoreTM. No antibody (black bars), MOR09823 (white bars), 105.5 (gray) and control IgG (dashed bars).
[0136] [00136] Figure 11: MOR09823-mediated inhibition of ligand-independent (BT-474) and (B) ligand-dependent (BxPC3) of HER3 in vivo.
[0137] [00137] Figure 12: The impact of MOR10701 and MOR10703 on the growth of the BT-474 tumor.
[0138] [00138] Figure 13: The impact of MOR10701 and MOR10703 on the growth of the BxPC3 tumor.
[0139] [00139] Figure 14: MOR10703 combination of the drug in isobolograms in vitro (A) MOR09823 / trastuzumab, (B) MOR09823 / lapatinib, (C) MOR10703 / BEZ235, (D) MOR10703 / BKM120, (E) MOR10703 / BYL719, (F) MOR10703 / RAD001, (G) MOR10703 cetuximab / e (H) MOR10703 / erlotinib.
[0140] [00140] Figure 15: MOR10701 MOR10703 or in in vivo combinations with (A) and trastuzumab (B) erlotinib in BT-474 and L3.3. Detailed Description of the Invention Definitions
[0141] [00141] In order that the present invention can be more easily understood, certain terms are defined first. Additional definitions are defined throughout the detailed description.
[0142] [00142] The phrase "signal transduction" or "signaling activity" as used in the present invention refers to a biochemical causal relationship usually initiated through a protein-protein interaction, such as the binding of a factor of growth to a receptor, which results in the transmission of a signal from one portion of a cell to another part of a cell. For HER3, transmission involves the specific phosphorylation of one or more tyrosine, serine or threonine in one or more proteins in the series of reactions that cause signal transduction. The penultimate processes typically include nuclear events, which result in a change in gene expression.
[0143] [00143] A "HER receptor" is a protein tyrosine kinase receptor that belongs to the HER receptor family and includes the EGFR, HER2, HER3 and HER4 receptors and other members of this family to be identified in the future. The HER receptor will generally comprise an extracellular domain, which can bind an HER ligand, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl terminal signaling domain harboring the various tyrosine residues that can be phosphorylated . Preferably, the HER receptor is a native sequence human HER receptor.
[0144] [00144] The terms "HER1," "ErbB1", "epidermal growth factor receptor" and "EGFR" are used interchangeably in the present invention and refer to EGFR as described, for example, in Carpenter et al . Ann. Rev. Biochem. 56: 881 to 914 (1987), including the naturally occurring mutant forms thereof (for example, a deletion mutant EGFR as in Humphrey et al., (1990) PNAS (US) 87: 4207 to 4211) . ErbB1 refers to the gene that encodes the EGFR protein product.
[0145] [00145] The terms "HER2" and "ErbB2" e are used in the present invention interchangeably and refer to the human HER2 protein described, for example, in Semba et al., (1985) PNAS (US) 82: 6497 to 6501 and Yamamoto et al. (1986) Nature 319: 230 to 234 (Genebank accession number X03363). The term "erbB2" refers to the gene encoding human ErbB2 and "neu" refers to the mouse encoding gene p185.
[0146] [00146] The terms "HER4" and "ErbB4" in the present invention refer to the polypeptide receptor as described, for example, in EP Patent No. Appln 599,274; Plowman et al, (1993) Proc .. Natl. Acad. U.S. Sci., 90: 1746 to 1750, and Plowman et al, (1993) Nature, 366: 473 to 475, including their isoforms, for example, described in WO99 / 19488, published on April 22, 1999.
[0147] [00147] The term "HER3" or "HER3 receptor" also known as "ErbB3" as used in the present invention refers to HER3 mammalian proteins and "HER3" or "erbB3" refers to the HER3 mammalian gene. The preferred HER3 protein is the human HER3 protein present in the cell membrane of a cell. The human HER3 gene is described in U.S. Patent No. 5,480,968 and Plowman et al., (1990) Proc. Natl. Acad. U.S. Sci., 87: 4905 to 4909.
[0148] [00148] Human HER3, as defined in Accession No. NP_001973 (human), hereinafter represented as SEQ ID NO: 1. All nomenclatures are for the full length, immature HER3 (amino acids 1 to 1342). Immature HER3 is cleaved between positions 19 and 20, which results in the mature protein HER3 (amino acids 20 to 1342). mrandalqvl gllfslargs evgnsqavcp gtlnglsvtg daenqyqtly klyercevvm gnleivltgh nadlsflqwi revtgyvlva mnefstlplp nlrvvrgtqv ydgkfaifvm Inyntnssha Irqlrltqlt eilsggvyie kndklchmdt idwrdivrdr daeivvkdng rscppchevc kgrcwgpgse dcqtltktic apqcnghcfg pnpnqcchde caggcsgpqd tdcfacrhfn dsgacvprcp qplvynkltf qlepnphtky qyggvcvasc phnfvvdqts cvracppdkm evdknglkmc epcgglcpka cegtgsgsrf qtvdssnidg fvnctkilgn ldflitglng dpwhkipald peklnvfrtv reitgylniq swpphmhnfs vfsnlttigg rslynrgfsl limknlnvts lgfrslkeis agriyisanr qlcyhhslnw tkvlrgptee rldikhnrpr rdcvaegkvc dplcssggcw gpgpgqclsc rnysrggvcv thcnflngep refaheaecf schpecqpme gtatcngsgs dtcaqcahfr dgphcvsscp hgvlgakgpi ykypdvqnec rpchenctqg ckgpelqdcl gqtlvligkt hltmaltvia glvvifmmlg gtflywrgrr iqnkramrry lergesiepl dpsekankvl arifketelr klkvlgsgvf gtvhkgvwip egesikipvc ikviedksgr qsfqavtdhm laigsldhah ivrllglcpg sslqlvtqyl plgslldhvr qhrgalgpql llnwgvqiak gmyyleehgm vhrnlaarnv llkspsqvqv adfgvadllp pddkqllyse aktpikwmal esihfgkyth qsdvwsygvt vwelmtfgae pyaglrlaev pdllekgerl aqpqictidv ymvmvkcwmi denirptfke laneftrmar dpprylvikr esgpgiapgp ephgltnkkl eevelepeld ldldleaeed nlatttlgsa lslpvgtlnr prgsqsllsp ssgympmnqg nlgescqesa vsgssercpr pvslhpmprg clasessegh vtgseaelqe kvsmcrsrsr srsprprgds ayhsqrhsll tpvtplsppg leeedvngyv mpdthlkgtp ssregtlssv glssvlgtee ededeeyeym nrrrrhspph pprpssleel gyeymdvgsd lsaslgstqs cplhpvpimp etiquetattpdedy eymnrqrdgg gpggdyaamg acpaseqgye emrafqgpgh qaphvhyarl ktlrsleatd safdnpdywh srlfpkanaq rt (SEQ ID NO: 1 )
[0149] [00149] The term "HER linker" as used in the present invention refers to polypeptides that bind to and activate HER receptors such as HER1, HER2, HER3 and HER4. Examples of HERS ligands include, but are not limited to, neuregulin 1 (NRG), neuregulin 2, neuregulin 3, neuregulin 4, betacellulin, heparin-binding epidermal growth factor, epiregulin, epidermal growth factor, amphiregulin and alpha factor transformation growth. The term includes biologically active fragments and / or variants of a naturally occurring polypeptide.
[0150] [00150] The term "HER3 linker", as used in the present invention, refers to the polypeptides that bind and activate HER3. Examples of HER3 ligands include, but are not limited to, neuregulin 1 (NRG) and neuregulin 2, betacellulin, heparin-binding epidermal growth factor, and epiregulin. The term includes biologically active fragments and / or variants of a naturally occurring polypeptide.
[0151] [00151] The "HER-HER protein complex" is a non-covalently associated oligomer containing the HER co-receptors in any combination (for example, HER1 - HER2, HER1 - HER3, HER1 - HER4, HER2 - HER3, HER3 - HER4 , and the like). This complex can form when a cell that expresses both receptors exposed to a ligand, for example HER, NRG, or when an HER receptor is active or overexpressed.
[0152] [00152] The "HER2-HER3 protein complex" is a non-covalently associated oligomer containing the HER2 receptor and the HER3 receptor. This complex can form when a cell that expresses both receptors exposed to a ligand, for example HER3, HER2 NRG or when it is active / overexpressed
[0153] [00153] The phrase "HER3 activity" or "HER3 activation", as used in the present invention refers to an increase in oligomerization (for example, an increase in complexes containing HER3), HER3 phosphorylation, conformational rearrangements (for example induced by the ligands), and the downstream-mediated HER3 signaling.
[0154] [00154] The term "stabilization" or "stabilized", used in the context of HER3 refers to an antibody or the fragment it directly maintains (locks, ties, maintainers, preferably, binders, favors) in the inactive or conformation state HER3, without blocking the binding of the ligand to HER3, a connection that such a ligand is no longer capable of activating HER3. The assays described in the examples can be used to measure the binding of the ligand to a stabilized HER3 receptor, for example, Biacore assay.
[0155] [00155] The term "ligand-dependent signaling" as used in the present invention refers to the activation of HER (e.g., HER3), through the ligand. HER3 activation is evidenced by increasing oligomerization (for example, heterodimerization) and / or HER3 phosphorylation in such a way that the downstream signaling pathways (for example, PI3K) are activated. The antibody or a fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in the stimulated cell exposed to the antigen-binding protein (for example, an antibody) relative to an untreated (control) cell, as measured using the assays described in the Examples. The cell that expresses HER3 can be a naturally occurring cell line (for example, MCF7), or it can be produced recombinantly by introducing nucleic acids encoding the HER3 protein into a host cell. Stimulation of cells can occur either through the exogenous addition of an HER3 activation ligand or through the endogenous expression of an activator ligand.
[0156] [00156] The antibody or a fragment thereof that "reduces neregulin-induced HER3 activation in a cell" is one that significantly reduces HER3 phosphorylation of tyrosine relative to an untreated (control) cell, as measured using the tests described in the Examples. This can be determined based on levels of phosphotyrosine HER3, after exposure of HER3 to NRG and the antibody of interest. The cell that expresses the HER3 protein can be a naturally occurring cell or cell line (for example, MCF7), or it can be produced recombinantly.
[0157] [00157] The term "ligand-independent signaling" as used in the present invention refers to cellular HER3 activity (e.g., phosphorylation), in the absence of a requirement for ligand binding. For example, the independent activation of the HER3 ligand may be the result of an overexpression of HER2 or activating mutations of HER3 partner heterodimers, such as EGFR and HER2. The antibody or a fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in a cell exposed to the antigen-binding protein (eg, an antibody) relative to an untreated (control) cell. The cell that expresses HER3 can be a naturally occurring cell line (for example, SK-BR-3), or it can be produced recombinantly by introducing nucleic acids encoding the HER3 protein into a host cell .
[0158] [00158] The term "blocking" as used in the present invention refers to containing or preventing an interaction or a process, for example, by stopping ligand-dependent or ligand-independent signaling.
[0159] [00159] The term "recognizes" as used in the present invention refers to an antibody or fragment thereof that locates and interacts (e.g., binds) with its conformational epitope.
[0160] [00160] The phrase "binds at the same time" used in the present invention refers to an HER linker that can bind to a linker binding site on the HER receptor along with the HER antibody. This means that both the antibody and antibody can bind to the HER receptor together. For the sake of illustration only, the HER3 NRG ligand can bind to the HER3 receptor, along with the HER3 antibody. Assay to measure the binding of the ligand simultaneously and of antibodies that are described in the examples section (for example, Biacore).
[0161] [00161] The term "failure" as used in the present invention refers to an antibody or fragment thereof that does not cause a particular event. For example, an antibody or fragment thereof that "does not activate signal transduction" is one that does not transduce the trigger signal, an antibody or fragment thereof that "fails to induce conformational change" is one that it does not cause a structural change in the HER receptor, an antibody or a fragment thereof, which stabilizes the HER receptor in an inactive state such that the HER receptor "fails to dimerize" is one that does not form protein-protein complexes.
[0162] [00162] The term "antibody" as used in the present invention refers to complete antibodies that interact with (for example, by steric impediment, binding, destabilizing spatial stabilization / distribution) an HER3 epitope and inhibit signal transduction. The naturally occurring "antibody" is a glycoprotein that comprises at least inter-chains of two heavy chains (H) and two light chains (L) linked by means of the disulfide bonds. Each heavy chain comprises a heavy chain variable region (in the present invention abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (in the present invention abbreviated as VL) and a light chain constant region. The light chain constant region consists of a CL domain. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, designated through structure regions (FR). Each VH and VL is composed of three CDRs and four FRs organized from the amino terminal to the carboxyl terminal, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the light and heavy chains contain a binding domain that interacts with an antigen. The antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classic complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single chain Fvs (scFv), disulfide bound to Fvs (sdFv), Fab fragments, F (ab ') fragments , and anti-idiotypic (anti-Id) (including antibodies, for example, anti-Id antibodies to antibodies of the present invention), and epitope-binding fragments of any of the above. The antibodies can be of any isotype (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
[0163] [00163] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be noted that the variable domains of the portions of both the light chain (VL) and the heavy chain (VH) determine antigen recognition and specificity. On the other hand, the light chain (CL) and heavy chain (CH1, CH2 or CH3) constant domains confer important biological properties such as secretion, transplacental mobility, binding to the Fc receptor, complement binding and the like. By convention, the numbering of the domains in the constant region increases as they become more distal from the antigen-binding site or amino terminal of the antibody. The N-terminal is a variable region and at the end of the C-terminal is a constant region, and the CH3 and CL domains actually comprise the carboxyl terminal of the light and heavy chain, respectively.
[0164] [00164] The term "antibody fragment", as used in the present invention, refers to one or more portions of an antibody that retain the ability to interact specifically with (for example, by means of steric impediment, binding, stabilization / destabilization, of the spatial distribution) an HER3 epitope and signal of inhibition of transduction. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by means of a disulfide bridge in the hinge region, an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al, (1989) Nature 341.: 544 to 546), consisting of a VH domain and a complementarity region isolated determinant (CDR).
[0165] [00165] Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by means of separate genes, they can be joined, using recombinant methods, by means of a synthetic linker that allows them to be made as a chain of single protein, in which the regions of VL and VH pairs have the purpose of forming monovalent molecules (known as single-chain Fv (scFv), see for example, Bird et al, (1988) Science 242: 423 to 426, and Huston et al, (1988) Proc Natl Acad. Sci. 85: 5879 to 5883). Such single chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same way as intact antibodies.
[0166] [00166] Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, tribodies, tetribodies, v-NAR and bis-scFv (see, for example, Hudson and Hollinger, (2005) Nature Biotechnology 23: 1126 to 1136). Antibody fragments can be grafted onto supports based on polypeptides such as Fibronectin type III (FN3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide antibodies).
[0167] [00167] The antibody fragments can be incorporated into single chain molecules that comprise a pair of tandem Fv segments (VH-CH1-VH-CH1) that, together with the complementary light chain polypeptides, form a pair of regions of antigen binding (Zapata et al., (1995) Protein Eng 8: 1057 to 1062;. and US Patent No. 5,641,870).
[0168] [00168] The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface clusters of molecules such as amino acids or carbohydrates or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope can be "linear" or "conformational".
[0169] [00169] The "linear epitope" refers to an epitope with all the points of interaction between the protein and the interacting molecule (such as an antibody) that occurs in a linear manner along the primary amino acid sequence of the protein (continuous) . Once the epitope on a desired antigen is determined, it is possible to generate antibodies against the epitope that, for example, uses the techniques described in the present invention. Alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information about desirable epitopes. From this information, it is then possible to competitively track antibodies for binding to the same epitope. One approach to achieve this goal is to conduct cross-competition studies to find antibodies that bind competitively with one another, for example, antibodies compete for antigen binding. A high-throughput process for binning antibodies based on their cross-competition is described in International Patent Application No. WO 2003 / 48731. As will be appreciated by one who is skilled in the art, practically nothing to which an antibody can be used. specifically linking to an epitope can be. An epitope can comprise the residues to which the antibody binds.
[0170] [00170] The "conformational epitope" refers to a discontinuous epitope in which the amino acids come together in three three-dimensional conformations. In a conformational epitope, points of interaction occur between the amino acid residues in the protein that are separated from each other. In one embodiment, the epitope is described in the Examples of the present invention. In one embodiment, the conformational epitope is defined by (i) amino acid residues HER3 265277 and 315 (from domain 2) and (ii) amino acid residues HER3 571.582584, 596-597, 600 to 602, 609 -615 ( 4) domain of SEQ ID NO: 1, or a subset of it. As will be appreciated by one skilled in the art, the space that is occupied by a side chain residue or that creates the shape of a molecule helps to determine whether an epitope.
[0171] [00171] Generally, antibodies specific to a particular target antigen preferably recognize an epitope on the target antigen from a complex mixture of proteins and / or macromolecules.
[0172] [00172] The regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Moris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined by, for example, concurrently synthesizing a large number of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and from reacting with the antibodies, the peptides, while the peptides are still attached to the supports. Such techniques are known in the art and are described in, for example, U.S. Patent No. 4,708,871; Geysen et al, (1984) Proc .. Natl. Acad. U.S. Sci. 8: 3998 to 4002; . Geysen et al, (1985) Proc. Natl. Acad. U.S. Sci. 82: 78 to 182; Geysen et al, (1986) Mol. Immunol. 23: 709 to 715. Likewise, conformational epitopes are readily identified by determining the spatial conformation of amino acids such as, for example, hydrogen / deuterium exchange, X-ray crystallography and two-dimensional magnetic resonance nuclear. See, for example, Epitope Mapping Protocols, supra. The antigenic regions of the proteins can also be identified using standard antigenicity and hydropathic plots, such as those calculated using, for example, the software program Omiga version 1.0 available from the Oxford Molecular Group. This computer program employs the method of Hopp / Woods, Hopp et al., (1981) Proc. Natl. Acad. Sci U.S. 78: 3824 to 3828, to determine antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al, (1982) J.MoI .. Biol. 157: 105 to 132, for hydropathy plots.
[0173] [00173] The term "paratope" as used in the present invention refers to the general structure of a binding region that determines binding to an epitope. This structure influences or not and how the binding region can bind to an epitope. Paratope can refer to an antigenic site of an antibody that is responsible for a fragment of the antibody, or, if it binds to an antigenic determinant. Paratope also refers to the antibody's idiotope, and the complementarity determining region (CDR), which binds to the epitope. In one embodiment, the paratope is the region of the antibody that binds to the conformational epitope comprising (i) amino acid residues HER3 265-277 and 315 (domain 2), and (ii) amino acid residues HER3 571, 582- 584, 596-597, 600 to 602, 609-615 (domain 4) of SEQ ID NO: 1, or a subset thereof. In one embodiment, the paratope is the region of the antibody that comprises the CDR sequences. In one embodiment, the paratope comprises the sequences shown in table 1. In one embodiment, the paratope comprises at least one amino acid residue that binds with the HER3 residues: Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276, Asn275, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597. In one embodiment, the paratope comprises at least one amino acid residue that binds with HER3 Residues: Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Lys602, Ile600, Arg611, Glu609, Pro612, Cys613, His614, Glu615. As will be appreciated by one skilled in the art, the paratope of an antibody, or a variant thereof, can be determined as established by the present application.
[0174] [00174] The phrases "monoclonal antibody" or "monoclonal antibody composition", as used in the present invention, refer to polypeptides, including antibodies, antibody fragments, bispecific antibodies, etc., which have substantially identical to the amino acid sequence or are derived from the same genetic source. This term also includes the preparation of antibody molecules of the single molecular composition. A monoclonal antibody composition has unique binding and affinity specificity for a particular epitope.
[0175] [00175] The phrase "human antibody", as used in the present invention, includes antibodies that have the variable regions of both regions that the structure and CDR are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region, it is also derived from human sequences such as, for example, human germline sequences, or mutant versions of human germline sequences or antibodies that contain consensus sequences derived from human structure from structure analysis in SEQuências, for example, as described in Knappik et al., (2000) J Mol Biol 296: 57 to 86). The structures and locations of the immunoglobulin variable domains, for example, CDRs, can be defined using well-known numbering systems, for example, the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, for example, Sequences of Proteins of Interest Immunology, US Department of Health and Human Services (1991), eds Kabat et al, Lazikani et al, (1997) J. Mol. Bio 273: 927 to 948), Kabat et al, (1991). Sequences of proteins of immunological interest, issue 5., NIH no publication. 91 to 3242 U.S. Department of Health and Human Services ,. Chothia et al, (1987) J. Mol. Biol. 196: 901 to 917; Chothia et al, (1989) Nature 342: 877-883; . And Al-Lazikani et al, (1997) J. Mol. Biol .. Biol. 273: 927 to 948.
[0176] [00176] The human antibodies of the present invention can include amino acid residues not encoded by means of human SEQUENCES (for example, mutations introduced by random mutagenesis or site-specific in vitro or by somatic mutation in vivo, or conservative substitution for the purpose of promoting stability or manufacturing).
[0177] [00177] The term "human monoclonal antibody", as used in the present invention, refers to antibodies having a unique binding specificity that have the variable regions in which both the framing and CDR regions are derived from human sequences. In one embodiment, human monoclonal antibodies are produced by means of a hybridoma that includes a B cell obtained from a non-human transgenic animal, for example, a transgenic mouse, having a genome comprising a human heavy chain transgene and a transgene of the light chain fused to an immortalized cell.
[0178] [00178] The phrase "recombinant human antibody", as used in the present invention, includes all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies isolated from an animal (for example, a mouse) which is transgenic or transcromosomal to human gene immunoglobulin or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, for example, from a transfectome, antibodies isolated from a library of recombinant, human combinatorial antibodies, and antibodies prepared, expressed, created or isolated by any other means that involve slicing all or a portion of a human immunoglobulin gene, the sequences for other DNA sequences. Such human recombinant antibodies have variable regions in which the framework and CDR regions are derived from immunoglobulin sequences of the human germline. In certain embodiments, however, such recombinant human antibodies may undergo muetiquetaesis in vitro (or, when an animal, transgenic to human Ig sequences when used, somatic muetequetaesis in vivo) and, therefore, the amino acid sequences of the regions VH and VL of recombinant antibodies are sequences that, although derived from and related to the human germline in SEH sequences VH and VL, may not naturally exist in the repertoire of the human germline of the antibody in vivo.
[0179] [00179] The specific connection between two entities means a connection with an equilibrium constant (KA) (kon / koff) of at least 102M-1, at least 5x102M-1, at least 103M-1, at least 5x103M-1, at least 104M-1at At least 5x104M-1, at least 105M-1, at least 5x105M-1, at least 106M-1, at least 5x106M-1, at least 107M-1, at least 5x107M-1, at least 108M- 1, at least 5x108M -1, at least 109M-1, at least 5x109M-1, at least 1010m-1, at least 5x1010M-1, at least 1011M-1, at least 5x1011M-1, at least 1012M-1 at least 5x1012M-1, at least 1013M-1, at least 5x1013 M-1, at least 1014M-1, at least 5x1014M-1, at least 1015M-1, or at least 5x1015M-1.
[0180] [00180] The phrase "specifically (or selectively) binds" to an antibody (for example, an HER3 binding antibody) refers to a binding reaction that is determinative of the presence of a related antigen (for example, a human HER3 ) in a heterogeneous population of proteins and other biological products. In addition to the equilibrium constant (KA) mentioned above, a HER3 binding antibody of the present invention typically also has a constant dissociation rate (KD) (koff / kon) of less than 5x10-2 M, less than 10-2M, less less than 5x10 -3M, less than 10-3M, less than 5x10-4 M, less than 10-4M, less than 5x10-5M, less than 10-5M, less than 5x10 to 6M, less than 10 to 6M, less at 5x10-7 M, less than 10-7M, less than 5x10-8 M, less than 10-8M, less than 5x10-9 M, less than 10-9M, less than 5x10-10 M, less than 10-10M , less than 5x10-11M, less than 10-11M, less than 5x10-12 M, less than 10-12M, less than 5x10-13M, less than 10-13M, less than 5x10-14M, less than 10-14M, less than 5x10-15M, or less than 10-15M or less, and binds to HER3 with an affinity that is at least twice as high as its affinity for binding to a non-specific antigen (eg, HSA) .
[0181] [00181] In one embodiment, the antibody or a fragment thereof has a dissociation constant (Kd) of less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than ten hours, less than 13:00, as as assessed by a method in the present invention described or known to one skilled in the art (for example, a BIAcore, ELISA, FACS, SET assay) (Biacore International AB, Uppsala, Sweden). The term "Kassoc" or "Ka", as used in the present invention, refers to the rate of association of a particular antibody-antigen interaction, while the term "Kdis" or "Kd", as used in present invention, refers to the rate of dissociation of a given antibody-antigen interaction. The term "KD", as used in the present invention, refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as the molar concentration ( M). Kd values for antibodies can be determined using methods well established in the art. One method for determining the KD of an antibody is by means of surface plasma resonance, or using a biosensor system, such as a Biacore ® system.
[0182] [00182] The term "affinity", as used in the present invention, refers to the force of interaction between the antibody and the antigen at individual antigenic sites. Within each antigenic site, the variable region of the "arm" antibody interacts through weak non-covalent forces with the antigen at numerous sites, the greater the interactions, the stronger the affinity.
[0183] [00183] The term "greed" as used in the present invention refers to an informative measure of overall stability or the strength of the antibody-antigen complex. It is controlled by three main factors: epitope affinity antibody, the valence of both antigen and the antibody, and the structural arrangement of the interacting parts. Ultimately, these factors define the specificity of the antibody, that is, the likelihood that the antibody is specific for an accurate antigen binding epitope.
[0184] [00184] The term "valence" as used in the present invention refers to the number of potential target sites for binding a polypeptide. Each target binding site specifically binds to a target molecule or specific site (i.e., epitopes) on a target molecule. When a polypeptide is composed of more than one target binding site, each target binding site can specifically bind to the same or different molecules (for example, it can bind to different molecules, for example, different antigens or epitopes, different molecules).
[0185] [00185] The phrase "anetiquetaonist antibody" as used in the present invention refers to an antibody that binds with HER3 and neutralizes the biological activity of HER3 signaling, for example, reduces, decreases and / or inhibits induced activity of HER3 signaling, for example, from a phosphor-HER3 or phospho-assay Akt. Test examples are described in more detail in the examples below. Accordingly, an antibody that "inhibits" one or more of these HER3 functional properties (for example, biochemical, immunochemical, cellular, physiological or other biological activity, or the like), as determined according to methodologies known in the art and described in present document, it will be understood to relate to a statistically significant decrease in specific activity relative to that observed in the absence of the antibody (for example, or when an irrelevant specificity control antibody is present). An antibody that inhibits the effects of HER3 activity such as a statistically significant decrease in at least 10% of the measured parameter, at least 50%, 80% or 90%, and in certain embodiments of an antibody of the present invention can inhibit more than than 95%, 98% or 99% of HER3 functional activity, as evidenced by a reduction in the level of cellular HER3 phosphorylation.
[0186] [00186] The phrase "isolated antibody" refers to an antibody that is substantially free of other antibodies that have different antigen specificities (for example, an isolated antibody that specifically binds to HER3 is substantially free of antibodies that specifically bind to other HER3 antigens). An isolated antibody that specifically binds toHER3 may, however, cross-react with other antigens. In addition, an isolated antibody can be substantially free of other cellular material and / or chemicals.
[0187] [00187] The term "modified conservative variant" applies to both amino acid and nucleic acid sequences. With regard to SE nucleic acid determinations, conservatively modified variants refer to nucleic acids encoding amino acid sequences, identical or essentially identical, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences . Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such variations of nucleic acids are "silent variations", which are a kind of conservatively modified variations. Each nucleic acid sequence in the present invention that encodes a polypeptide also describes any possible silent variation of the nucleic acid. A person skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the codon for methionine only, and TGG, which is normally the codon for tryptophan only) can be modified to produce a functionally identical molecule. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.
[0188] [00188] For polypeptide sequences, "conservatively modified variants" includes individual substitutions, deletions or additions to a polypeptide sequence that results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that functionally provide similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, homologous between species, and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); Asparagine 3) (N), Glutamine (, 2) Aspartic acid (D), Glutamic acid (E) Q), and 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W), 7) Serine (S), Threonine (T) , and 8) cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term "conservative modification sequence" is used to refer to amino acid modifications that do not affect or alter the binding characteristics of the antibody, containing the amino acid sequence.
[0189] [00189] The terms "cross competition" and "cross competitor" are used in the present invention interchangeably to refer to the ability of an antibody or other binding agent to interfere with the binding of antibodies or other binding agents to HER3 in a competitive standard bond assay.
[0190] [00190] The ability of an antibody or other binding agent is capable of interfering with the binding of another antibody or binding molecule to HER3, and therefore, whether it can be said to cross-compete according to the present invention, can be determined using standard application binding tests. A suitable test involves the use of Biacore technology (for example, using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of the interaction using surface plasmon resonance technology. Another test to measure cross-competition uses an ELISA-based approach.
[0191] [00191] The term "optimized" as used in the present invention refers to a nucleotide sequence that has been altered for the purpose of encoding a sequence of amino acids using codons that are preferred in the production of cells or organism, in general, a eukaryotic cell, for example, a Chi cell, a Trichoderma cell, a Chinese hamster ovary (CHO) cell, or a human cell. The nucleotide sequence is optimized to retain as much or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.
[0192] [00192] Standard assays to assess the binding capacity of antibodies to HER3 of various species are known in the art, including for example, ELISAs, RIAs and western blots. Suitable tests are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies can also be assessed by means of standard assays known in the art, such as by means of Biacore analysis, or FACS relative affinity (Scatchard). Assays to assess the effects of antibodies on the functional properties of the HER3 assays (e.g., binding receptor, via its modulator) are described in more detail in the Examples.
[0193] [00193] The phrases "identical percent" or "percent identity", in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percent of amino acid or nucleotide residues that are the same (i.e., 60% identity, optionally, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a given region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or region designated as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more acidic amino acids) in length.
[0194] [00194] For sequence comparison, typically a sequence acts as a reference sequence, in which the test sequences are compared. When using a test sequence comparison algorithm, and the reference sequences are entered into a computer, coordinated subsequences are designated, if necessary, and the program parameters of the sequence algorithm are designated. Standard program parameters can be used, or alternate parameters can be assigned. The sequence comparison algorithm then calculates the percentage of sequence identities for the test sequences in relation to the reference sequence, based on the program parameters.
[0195] [00195] A "comparison window", as used in the present invention, includes reference to a segment of any of several contiguous positions selected from the group consisting of from 20 to 600, generally about 50 to about 200 , more usually about 100 to about 150, in which a sequence can be compared with a reference sequence of the same number of contiguous positions, after the two sequences have been optimally aligned. Sequence alignment methods for comparison are well known in the art. The optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2: 482 c, using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. Biol. 48: 443, through the search for the similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444, through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI), or through manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).
[0196] [00196] Two examples of algorithms that are suitable for determining the sequence identity and sequence similarity tag are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25: 3389 to 3402 and Altschul et al, (1990) J. Mol. Biol .. Biol. 215: 403 to 410, respectively. The software for performing BLAST analyzes is publicly available through the National Biotechnology Information Center. This algorithm first involves identifying high-sequence punctuation pairs (HSPs), identifying short words of length W in the query string, which may come to match or satisfy some positive values with T threshold when aligned with a word of the same length in a database string. T is referred to as the scoring limit for neighboring words (Altschul et al., Supra). These initial neighboring words act as seeds to initiate searches to find more HSPs that contain them. Word hits are extended in both directions throughout each sequence, as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always> 0) and N (penalty score for decoupling residues; always <0). For amino acid sequences, a classification matrix is used to calculate the cumulative score. The extension of word accesses in each direction is interrupted when: the cumulative alignment score falls by the quantity X from its maximum value reached; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments, or the end of any sequence is reached. The parameters of the BLAST algorithm W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defects a word length (W) of 11, an expectation (E) or 10, M = 5, N = -4 and a comparison of both strings. For amino acid sequences, the BLASTP program defects a word length of 3, and expectation (E) of 10, and the BLOSUM62 punctuation matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sei. US 89: 10915) alignments (B) of 50, expectation (E) of 10, M = 5, N = -4 and a comparison of both chains.
[0197] [00197] The BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, for example, Karlin and Altschul, Proc (1993). Natl. Acad. Sci. U.S. 90: 5873 to 5787). A measure of the similarity provided by the BLAST algorithm is the probability of the smallest sum (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the probability of the smallest sum in a comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and more preferably less than about 0.001.
[0198] [00198] The identity tag between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (1988) Comput. Appl. Biosci. 4: 11 to 17), which was incorporated into the ALIGN program (version 2.0), using a weight table PAM120 of weight, a gap length penalty of 12 and a gap penalty of 4. In addition, the identity tag between the two amino acid sequences can be determined using Needleman and Wunsch (1970) J. Mol. Biol. Biol. 48: 444a453) the algorithm that was incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a weight of 1,2, 3, 4, 5, or 6 in length.
[0199] [00199] Except for the identity tag of the sequence mentioned above, another indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies created against the encoded polypeptide by the second nucleic acid, as described below. In this way, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only through conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
[0200] [00200] The phrase "nucleic acid" is used in the present invention interchangeably with the term "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double stranded form. The term encompasses nucleic acids containing the known nucleotide analogs or modified residues or bonds of main structure, which are synthetic, naturally occurring, and which occur unnaturally, which have similar binding properties, such as nucleic acid reference cells and are metabolized in a similar way to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl-ribonucleotide (PNA) nucleic acid peptide.
[0201] [00201] Unless otherwise used, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Degenerate codon substitutions specifically, as detailed below, can be achieved by generating sequences in which the third position of one or more selected codons (or all) is replaced with mixed base residues and / or deoxyinosine (Batzer et al ., (1991) Nucleic Acid Res. 19: 5081 ,. Ohtsuka et al, (1985) J. Biol Chem 260: 2605 to 2608, and Rossolini et al, (1994) Mol. Cell Probes 8: 91 to 98).
[0202] [00202] The phrase "operationally linked" refers to a functional relationship between two or more polynucleotide segments (for example, DNA). Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence that stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, transcription promoters that are regulatory sequences operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, that is, they are cis-reached. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically located contiguously or in close proximity to the coding sequences whose transcription improves it.
[0203] [00203] The terms "polypeptide" and "protein" are used interchangeably in the present invention to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of an amino acid that occurs in a corresponding natural way, as well as the non-natural amino acid polymers of the amino acid polymer that occur in a Natural. Unless used in another way, a given polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
[0204] [00204] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Except where otherwise indicated, the terms "patient" or "subject" are used in the present invention interchangeably.
[0205] [00205] The term "anti-cancer agent" means any agent that can be used to treat a disorder of cell proliferation such as cancer, including cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents and agents immunotherapeutic.
[0206] [00206] The term "tumor" refers to the growth of neoplastic and proliferating cells, whether malignant or benign, and all precancerous and cancerous cells and tissues.
[0207] [00207] The term "anti-tumor activity" means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity. One possible way to show anti-tumor activity shows a decline in the rate of growth of abnormal cells that arise during treatment or the stability of the tumor size or reduction. Such activity can be evaluated using accepted in vitro or in vivo tumor models, including, but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.
[0208] [00208] The "malignant neoplasm" refers to a benign tumor or non-cancer. As used in the present invention, the term "cancer" includes a malignancy characterized by unregulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas. The term "cancer" includes primary malignant tumors (for example, those in which cells do not migrate to locations on the subject's body other than the original tumor site) and secondary malignant tumors (for example, those from metastases, migration tumor cells to secondary sites, which are different from the original tumor site).
[0209] [00209] Various aspects of the present invention are described in more detail in the following sections and subsections. Structure and mechanism of activation of HER receptors
[0210] [00210] All four HER receptors have an extracellular ligand binding domain, a single transmembrane domain, and a domain containing cytoplasmic tyrosine kinase. The intracellular tyrosine kinase domain of HER receptors is highly conserved, although the HER3 kinase domain contains critical amino acid substitutions and therefore lacks kinase activity (Guy et al, (1994): PNAS 91, 8132 a 8136). HER receptor ligand-induced dimerization induces kinase activation, receptor transfosphorylation in tyrosine residues at the C-terminal tail, followed by recruitment and activation of intracellular signaling effectors (Yarden and Sliwkowski, (2001) Nature Rev 2, 127 to 137; Jorissen et al, (2003) Exp. Cell Res. 284, 31 to 53.
[0211] [00211] The crystalline structures of the HERS extracellular domains have provided some clarification about the activation process induced by the receptor ligand (Schlessinger, (2002) Cell 110, 669 to 672). The extracellular domain of the HER receptor each consists of four subdomains: subdomain I and III cooperate in the formation of the ligand binding site, while subdomain II (and perhaps also subdomain IV) participates in the dimerization of the receptor via direct receptor-receptor interactions. . In ligand structures linked to HER1, a β clamp (called the dimerization loop) from subdomain II interacts with the dimerization circuit of the partner receptor, mediating the dimerization of the receptor (Garrett et al, (2002) Cell 110, 763 to 773 ; Ogiso et al., (2002) Cell 110, 775 to 787). In contrast, in the structures of HER1, HER3 and inactive HER4, the dimerization loop is involved in intramolecular interactions with subdomain IV, which prevents the dimerization of the receptor in the absence of ligand (Cho and Leahy, (2002) Science 297, 133 to 1333; Ferguson et al, (2003) Mol Cell 12, 541 to 552, Bouyan et al, (2005) PNAS102, 15024 to 15029). The structure of HER2 is unique among hers. In the absence of a ligand, HER2 has a conformation that resembles the activated ligand state of HER1 with a protruding dimerization loop, available to interact with other HER receptors (Cho et al, (2003) Nature 421, 756 to 760 ,. Garrett et al., (2003) Mol Cell 11, 495 to 505). This may explain the improved heterodimerization capacity of HER2.
[0212] [00212] Although the crystal structures of the HER receptor provide a model for the homo- and heterodimerization of the HER receptor, the backdrop for the prevalence of some HER homo and heterodimers to the detriment of others (Franklin et al., (2004) Cancer Cell 5, 317-328) as well as the conformational role of each of the domains in receptor dimerization and self-inhibition (Burgess et al, (2003) Mol. Cell 12, 541-552, .. Mattoon et al, (2004 ) PNAS101, 923-928) remains unclear. As described below, HER3 X-ray crystalline structure provides one more internal views. HER3 structure and conformational epitopes
[0213] [00213] A conformational epitope to which proteins bind to the antigen, for example, anti-HER3 binding antibodies, is provided in the present invention. For the first time, the three-dimensional structure of a truncated form (residues 20 to 640) of the extracellular domain of HER3 complexed with an antibody has been shown. The HER3-Fab MOR09823 complex and HER3-MOR09825 were determined at a resolution of 3.2a and 3.4 Å, respectively, and shown in Figure 5A. The present invention also demonstrates for the first time an antibody or a fragment thereof that binds to an inactive state of HER3 and stabilizes the receptor in an inactive state. The antibodies of the present invention also allow simultaneous binding of an HER3 ligand, such as neuregulin, with the HER3 receptor.
[0214] [00214] Although not required to provide a theory, a possible model for the mechanism of action is that normally HER3 exists in an inactive (closed, tied) or active (open) state. The ligand binding induces a conformational change that such HER3 exists in the active (open) state which is capable of heterodimer binding partners, resulting in activation of downstream signaling. Antibodies like MOR09823 bind the HER3 tethered state but do not block the ligand binding site. Antibodies, such as MOR09823 inhibit HER3, preventing the induced ligand structural rearrangements necessary for HER3 to transition to active conformation, thus preventing signal transduction. In one embodiment, the antibodies of the present invention or fragments thereof bind to the inactive state (with strings) of HER3, but do not block the ligand binding site. In another embodiment, the antibodies or their fragments inhibit HER3, preventing the structurally induced ligand rearrangements necessary for HER3 to transition to active conformation, thus preventing signal transduction. In another embodiment, the antibody or a fragment of it stabilizes (directly maintains, locks, ties, preferably maintains, binds, or favors) the HER3 receptor in an inactive or conformation state. In one embodiment, the inactive HER3 receptor may be susceptible to internalization or preferential degradation in such a way that it leads to the loss of HER3 cell surface receptors. The biological data presented in the Examples section supports these modalities.
[0215] [00215] HER3 crystals can be prepared by expressing a nucleotide sequence encoding HER3 or a variant thereof in a suitable host cell, and then crystallizing the purified protein (s) in the presence of the Fab Relevant HER3 targets.
[0216] [00216] HER3 polypeptides can also be produced as fusion proteins, for example to assist in extraction and purification. Examples of fusion partners include the protein glutathione-S-transferase (GST), histidine (HIS), hexa-histidine (6HIS), GAL4 ligand (DNA and / or transcriptional activation domains) and beta-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest in order to allow removal of fusion protein sequences.
[0217] [00217] After expression, proteins can be purified and / or concentrated, for example, by means of affinity chromatography with immobilized metal, ion exchange chromatography, and / or gel filtration.
[0218] The protein (s) can be crystallized using the techniques described in the present invention. Generally, in a crystallization process, containing a drop of the protein solution is mixed with the crystallization buffer and left to equilibrate in a sealed container. The balance can be achieved by means of known techniques, such as the "drop in suspension" or "dropping" method. In these methods, the drop is hung above or seated next to a much larger reservoir of the crystallization buffer and equilibrium is achieved by means of vapor diffusion. Alternatively, equilibrium can occur through other methods, for example, under oil, through a semi-permeable membrane, or through interface-free diffusion (See, for example, Chayen et al, Methods (2008) Nature, 147 to 153.
[0219] [00219] Once the crystals have been obtained, the structure can be resolved using known X-ray diffraction techniques. Many techniques use chemically modified crystals, such as those modified by means of heavy atom derivatization for the approximate phases. In practice, a crystal is embedded in a solution containing the salts of heavy metals of atoms, or organometallic compounds, chloride, for example, lead, gold thiomalate, thimerosal or uranyl acetate, which can diffuse through the crystal and bind on the surface of the protein. The location (s) of the attached heavy metal atom (s) can then be determined by means of X-ray diffraction analysis of the embedded crystal. The patterns obtained in the diffraction of a monochromatic X-ray beam by the atoms (dispersion centers) of the crystal can be solved by means of mathematical equations to give mathematical coordinates. The diffraction data is used to calculate a map of the electronic density of the crystal repetitive unit. Another method of obtaining phase information is to use a technique known as molecular substitution. In this method, the rotation and translation algorithms are applied to a research model derived from a related structure, which results in an approximate orientation for the protein of interest (see Rossmann, (1990) Acta Crystals A 46, 73 to 82) . Electronic density maps are used to establish the positions of individual atoms within the unit cell of the crystal (Blundel et al., (1976) Protein Crystallography, Academic Press).
[0220] [00220] This specification describes, for the first time, the three-dimensional structure of HER3 and a Fab of an anti-HER3 antibody. The approximate domain limits of the HER3 extracellular domain are as follows; domain 1: 20 to 207 amino acids; domain 2: 208 to 328 amino acids; domain 3: 329 to 498 amino acids, and domain 4: 499 to 642 amino acids. The three-dimensional structure of HER3 and the antibody also allows the identification of target binding sites for potential HER3 modulators. The preferred target sites of binding are those involved in the activation of HER3. In one embodiment, the target binding site is located within domain 2 and domain 4 of HER3. In this way, an antibody or fragment that binds to either domain 2 or domain 4, and preferably from both domains is capable of modulating activation by HER3 or preventing the domains from dissociating from each other or by changing positions relative domains. In this way, the binding of an antibody, or a fragment thereof, to the amino acid residues within domain 2 or domain 4 can cause the protein to adopt a conformation that prevents activation. The present invention also demonstrates for the first time an antibody or a fragment thereof, which can simultaneously bind with an HER3 ligand, such as neuregulin.
[0221] [00221] In some embodiments, the antibody or a fragment thereof recognizes a specific HER3 conformation state such that the antibody or fragment thereof prevents HER3 from interacting with a co-receptor (including, but not limited to, HER1, HER2 and HER4). In some embodiments, the antibody or fragment thereof prevents HER3 from interacting with a co-receptor, stabilizing the HER3 receptor in an inactive or closed state. In one embodiment, the antibody, or a fragment thereof, stabilizes the HER3 receptor by binding to amino acid residues within domain 2 and domain 4 of HER3. In this inactive state, the dimerization circuit located within domain 2, is not exposed and, therefore, available for dimerization with other co-receptors (including, but not limited to, HER1, HER2 and HER4). In some embodiments, the antibody or a fragment thereof binds to the human HER3 protein having a conformational epitope comprising (i) the 265 to 277 amino acid residues HER3 and 315 (from domain 2) and (ii) 571 HER3 residues from amino acids, 582 to 584, 596 to 597, 600 to 602, 609 to 615 (domain 4) of SEQ ID NO: 1, or a subset thereof. In some embodiments, the antibody or a fragment thereof binds to amino acids within or overlapping the 265 to 277 amino acid residues and 315 (from domain 2) and (ii) 571 HER3 amino acid residues, 582 to 584, 596 to 597 , 600 to 602, 609 to 615 (domain 4) of SEQ ID NO: 1. In some embodiments, the antibody or a fragment thereof binds to amino acids within (and / or amino acid sequences consisting of) the 265 to 277 amino acid residues and 315 (from domain 2) and (ii) 571 HER3 amino acid residues, 582 to 584, 596 to 597, 600 to 602, 609 to 615 (domain 4) of SEQ ID NO: 1, or one subset of it. In some embodiments, the antibody or a fragment of it binds to the conformational epitope such that it restricts the mobility of domain 2 and domain 4, stabilizing it in an inactive or closed conformation. The inability to form the conformation result activates the inability to activate signal transduction. In some embodiments, the antibody or a fragment thereof binds to the conformational epitope in such a way that the dimerization circuit occludes within domain 2, making it available for receptor-receptor interaction. The inability to form homo- or heterodimers results in an inability to activate signal transduction.
[0222] [00222] In another aspect, the antibody or a fragment thereof binds to a conformational epitope of the HER receptor, such as an HER3 receptor. In one embodiment, the antibody or a fragment thereof, stabilizes the HER3 receptor, in an inactive state. In another embodiment, the antibody or a fragment thereof binds to the active state of the HER3 receptor and directs it in the inactive state as the inactive state. In this way, the antibody or a fragment of it can bind to either the active or inactive state of HER3, but it favors the formation of the inactive state and drives the active state of HER3 to the inactive state, which results in an inability to activate signal transduction.
[0223] [00223] In another aspect, the antibody or a fragment of it binds to a conformational epitope of the HER receptor, such as a HER3 receptor, where the binding of the antibody or a fragment thereof, stabilizes the HER3 receptor in an inactive state such that the HER3 receptor fails to dimerize with a co-receptor, in order to form a receptor-receptor complex. The inability to form a receptor-receptor complex prevents the activation of signal transduction, both dependent on the ligand and independent of the ligand.
[0224] [00224] In another aspect, the antibody or a fragment thereof binds to a conformational epitope of the HER receptor such as the HER3 receptor, wherein binding of the antibody or fragment thereof to the HER3 receptor allows dimerization with a coreceptor for the purpose of forming an inactive receptor-receptor complex. The formation of the receptor-receptor complex prevents the inactive activation of ligand-independent signal transduction. For example, ligand-independent signal transduction, HER3 may exist in an inactive state, however, overexpression of HER2 causes HER2 -HER3 to form the complex, however, these resulting complexes are inactive and prevent activation of ligand-independent signal transduction.
[0225] [00225] The structure described also allows the identification of HER3 amino acid residues specific to the nucleus for the interface of interaction of a fragment of the same antibody, or (for example, MOR09823) with HER3. This was defined as the residues that are within the 5A of the MOR09823 protein VH chain. The core residues are as follows: Asn266, Lys267, Leu268, Thr269, Gln271, Glu273, Pro274, Asn275, Pro276, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597.
[0226] [00226] The structures can also be used to identify the limits of the HER3 Residues of amino acids for the interface of interaction with an antibody or a fragment of it (for example, MOR09823). These residues can be HER3 residues that were 5 to 8A of the VH MOR09823 protein chain. The contour residues are as follows: Pro262, Val264, Tyr265, Phe270, Leu272, Thr278, Lys314, Gly316, Glu321, Asn566, Ser568, Gly569, Ser570, Thr572, Arg580, Asp581, Gly582, Gly595, Gly598, I600
[0227] [00227] The structure described also allows the identification of HER3 amino acid residues specific to the nucleus for the interface of interaction of a fragment of the same antibody, or (for example, MOR09823) with HER3. This was defined as the residues that are found within 5a of the V0 chain of the MOR09823 protein. The core residues are as follows: Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614, Glu615.
[0228] [00228] The structures were also used to identify the limits of the HER3 residues of amino acids for the interface of interaction with an antibody or a fragment of it (for example, MOR09823). These residues were Residues HER3 that were 5 to 8A of the MOR09823 protein VL chain. The contour residues are as follows: Asn266, Thr269, Asp571, Arg580, Asp581, His584, Pro590, Ala596, Pro599, Tyr601, Tyr603, Asp605, Gln607, Cys610, Asn616, Cys617, Cys621, Gly623, Pro624.
[0229] [00229] As can be seen in Tables 11 and 12 (MOR09823) and Tables 13 and 14 (MOR09825), respectively, the heavy chain is mainly involved in the binding protein antigen binding to amino acid residues within domain 2 of the epitope with less interactions with domain 4 amino acid residues, while the light chain is mainly involved with binding to amino acid residues within domain 4 of interactions with the epitope with less amino acid residues within domain 2.
[0230] [00230] As such, one skilled in the art, taking into account the present teachings, can predict that the residues and surfaces of the antigen-binding proteins can be varied without unduly interfering with the ability of the antigen-binding protein to bind to HER3.
[0231] [00231] The essential amino acids interacting at the amino acid interface were determined to be all amino acid residues with at least one atom less than or equal to 5-a from the HER3 partner protein. 5A was chosen as the core cutting distance to allow the region of atoms within a van der Waals radius plus a possible water-mediated hydrogen bond. The amino acids at the interaction frontier interface were determined to be all amino acid residues with at least one atom less than or equal to 8-A from the HER3 partner protein, but not included in the core interaction list.
[0232] [00232] In some embodiments, any antigen binding protein that binds to, caps on, or prevents MOR09823 from interacting with any of the above residues can be used to bind or neutralize HER3. In some embodiments, the antibodies or fragments thereof bind to or interact with at least one of the following HER3 residues (SEQ ID NO: 1): Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276 , Asn275, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597. In some embodiments, the antibodies and fragments thereof bind to or interact with at least one of the following HER3 residues (SEQ ID NO: 1): Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Lys602, Ile600, Glu609, Arg611, Pro612, Cys613, His614, Glu615. In some embodiments, the antibodies or fragments thereof bind to or interact with at least one of the following HER3 residues (SEQ ID NO: 1): Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276 , Asn275, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597, Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, C6 In some embodiments, the antibodies or fragments thereof bind to or interact with a combination of the following HER3 residues (SEQ ID NO: 1): Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276, Asn275, His277 , Asn315, Asp571, Pro583, His584, Ala596, Lys597, Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614, Glu615. In some embodiments, the antibodies or fragments thereof bind to or interact with all of the following HER3 residues (SEQ ID NO: 1): Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276, Asn275, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597, Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Ile600, Lys602, Glu609, Arg611, Pro612, Cys613, His614, Glu6. In some embodiments, the antibody or fragment thereof is 5 angstroms, from one or more of the aforementioned residues. In some embodiments, the antibody or a fragment thereof is 5 to 8 angstroms from one or more of the aforementioned residues. In some embodiments, the antibody or a fragment of it interacts, blocks, that is, within 8 angstroms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 of the above residues.
[0233] [00233] The availability of 3D structures of HER3 and the HER3 complex: MOR09823, for example, provides the framework for exploring other HER3 antibodies in more detail. The 3D structure of HER3 allows the epitopes for monoclonal antibodies to be mapped and their mode of action inferred, since some inhibit, stimulate some and others have no effect on cell growth. The conformational epitope of MOR09823 was found for domains 2 and 4 of HER3. The availability of the 3D structures of this receptor will facilitate the determination of the precise mechanism of action of these inhibitory agents and the design of new approaches to interfere with the function of the HER3 receptor. In one embodiment, the antibodies of the present invention bind to the same conformational epitope as MOR09823.
[0234] [00234] In some embodiments, the conformational epitope attached via any of the antibodies listed in Table 1 is especially useful. In certain embodiments, a HER3 conformational epitope can be used to isolate antibodies from binding fragments than HER3. In certain embodiments, a HER3 conformational epitope can be used to generate the antibodies or fragments thereof that bind to HER3. In certain embodiments, a HER3 conformational epitope can be used as an immunogen to produce antibodies to fragments that bind to the HER3 conformational epitope. In certain embodiments, a HER3 conformational epitope can be administered to an animal, and antibodies that bind to HER3 can subsequently be obtained from the animal.
[0235] [00235] In some modalities, the domain (s) / region (s) that contain the residues that are in contact with or are buried by means of an antibody can be identified by mutating specific residues in HER3 (for example, example, a wild-type antigen) and determine whether the antibody or fragment thereof can bind the HER3 mutants or variant of the protein or measure the differences in wild-type affinity. By making a series of individual mutations, residues that play a direct role in binding or that are in close enough proximity that the antibody in such a way that the mutation can affect the binding between the antibody and the antigen can be identified. From a knowledge of these amino acids, the domain (s) or region (s) of the antigen (HER3), which contains the residues in contact with the antibody or covered by the antibody can be elucidated. Muetiquetaênese using known techniques, such as alanine scanning, can help to define functionally relevant epitopes. Muetiquetaênese using a scanning arginine / glutamic acid protocol can also be employed (see, for example, Nanevicz et al., (1995), J. Biol. Chem. 270 (37): 21619 to 21625 and Zupnick et al. , (2006), J. Biol. Chem. 281 (29): 20464 to 20473). In general, arginine and glutamic acid are substituted (typically individually) for an amino acid in the wild-type polypeptide, as these amino acids are loaded and bulky and therefore have the potential to destroy the link between an antigen-binding protein and an antigen of the antigen region into which the mutation is introduced. Arginines that exist in the wild type antigen are replaced with glutamic acid. A variety of such individual mutants can be obtained and the binding results collected and analyzed to determine which residues affect the binding. A series of mutants of HER3 antigens can be created, with each mutant antigen having a single mutation. The binding of each mutant HER3 antigen to several HER3 antibodies or fragments thereof can be measured and compared with the ability of the selected antibody to one or the fragments thereof bind to wild-type HER3 (SEQ ID NO: 1).
[0236] [00236] A change (for example, a reduction or increase) in binding between an antibody or fragment thereof and a mutant or variant HER3, as used in the present invention, means that there is a change in binding affinity (for example , as measured by known methods, such as the Biacore test or assay based on the granule described below in the examples), EC50, and / or a change (for example, a reduction) in the total binding capacity of the binding protein to antigen (for example, as evidenced by a decrease in Bmax in an antigen binding portion in protein concentration as a function of antigen concentration). A significant change in binding indicates that the mutated residue is involved in binding the antibody or fragment thereof.
[0237] [00237] In some embodiments, a significant reduction in binding medium, that the binding affinity, EC50, and / or the capacity between an antibody, or fragments thereof, and a mutant HER3 antigen is reduced by more than 10 %, greater than 20%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85 %, greater than 90% or greater than 95% with respect to the binding between the antibody or fragment an HER3 and a wild type (for example, NO, SEQ ID NO: 1).
[0238] [00238] In some embodiments, the binding of an antibody or fragments thereof is significantly reduced or increased for a mutant HER3 protein that has one or more (for example, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mutations compared to a wild-type HER3 protein (for example, NO, SEQ ID NO: 1).
[0239] [00239] Although the variant forms are referenced in relation to the wild type sequence shown in SEQ ID NO: 1, it will be appreciated that, in the allelic variants of HER3 slicing the amino acids may differ. Antibodies or fragments thereof that show significantly altered binding (e.g., lower or higher binding) to such allelic forms of HER3 are also contemplated.
[0240] [00240] In addition to the general structural aspects of antibodies, a more specific interaction between the paratope and the epitope can be examined through structural approaches. In one embodiment, the structure of the CDRs contributes to a paratope, through which an antibody is able to bind to an epitope. The shape of such a paratope can be determined in a number of ways. Traditional structural examination approaches can be used, such as NMR or x-ray crystallography. These approaches can examine the shape of the paratope alone, or while it is attached to the epitope. Alternatively, molecular models can be generated in silico. The structure can be generated through homology modeling, with the help of a commercial packaging, as an InsightII Accelrys modeling package (San Diego, Calif.) In short, the antibody sequence to be analyzed for research against a database of proteins of known structures, such as the Protein Data Bank. After identification of homologous proteins with known structures, these homologous proteins are used as modeling models. Each of the possible models can be aligned, thus producing the alignments of base structure sequences between the molds. The antibody sequence with the unknown structure can then be aligned with these models in order to generate a molecular model of the antibody with the unknown structure. As will be appreciated by one skilled in the art, there are many alternative methods for producing such structures in silico, any of which can be used. For example, a process similar to that described in Hardman et al., Issued US Patent No. 5,958,708 QUANTA employing (polygen Corp, Waltham, Massachusetts) and CHARM (Brooks et al., (1983), J. Comp. Chem. 4: 187) can be used (in the present invention incorporated in its entirety by reference).
[0241] [00241] Not only is the form of the paratope important in determining whether and how a possible paratope will bind to an epitope, but the very interaction between the epitope and the paratope is a source of great information in the design of variant antibodies. As appreciated by one skilled in the art, there are a variety of ways in which this interaction can be studied. One way is to use the generated structural model, perhaps, as described above, and then use a program like InsightII (Accelrys, San Diego, California), which has a plug-in module, which, among other things, is capable of conducting a Monte Carlo research on the conformational and orientational spaces between the paratope and its epitope. The result is that the person is able to estimate where and how the epitope interacts with the paratope. In one embodiment, only a fragment, or variant, of the epitope is used to help determine the relevant interactions. In one embodiment, the entire epitope is used to model the interaction between the paratope and the epitope.
[0242] [00242] Through the use of these modeled structures, one is able to predict which residues are the most important in the interaction between the epitope and the paratope. Thus, in one embodiment, it is able to easily choose which residues to change in order to change the binding characteristics of the antibody. For example, it may be evident from the plug-in models that the side chains of certain residues in the paratope can sterically prevent the epitope from binding, changing these residues to residues with smaller side chains that may be beneficial. This can be determined in many ways. For example, one can simply look at the two models and interactions based on functional and proximity groups. Alternatively, repeated epitope and paratope pairings can be performed, as described above, in order to obtain the most favorable energy exchanges. These interactions can also be determined for a variety of antibody variants to determine alternative ways in which the antibody can bind to the epitope. One can also combine the various models to determine how to change the structure of the antibodies in order to obtain an antibody with the particular characteristics that are desired.
[0243] [00243] The models determined above can be tested using various techniques. For example, the interaction energy can determine with the programs discussed above, in order to determine which of the variants to continue to analyze. In addition, coulumbic and van der Waals interactions are used to determine the interaction energies of the epitope and the variant paratopes. Also, targeted muetiquetaênese is used to verify whether it predicted changes in the structure of the antibody, actually causing the desired changes in binding characteristics. Alternatively, changes can be made to the epitope to verify that the models are correct or to determine the general themes of connection that may be occurring between the paratope and the epitope.
[0244] [00244] As will be appreciated by one skilled in the art, while these models will provide the necessary guidance for making the antibodies and their variants of the present modalities, it may still be desirable to perform routine tests of the models in silico, perhaps through In vitro studies. In addition, as will be apparent to one skilled in the art, any modification may also have additional side effects on the activity of the antibody. For example, while no changes predicted to result in greater binding, can induce greater binding, it can also cause other structural changes that may reduce or alter the activity of the antibody. Determining whether or not this is the case is routine in the art and can be achieved in a number of ways. For example, the activity can be tested using an ELISA test. Alternatively, samples can be tested using a surface plasmon resonance device. HER3 Antibodies
[0245] [00245] The present invention provides antibodies that recognize a conformational epitope of HER3. The present invention is based on the surprising discovery that a class of antibodies against HER3, blocks both ligand-dependent and HER3-ligand-independent signal transduction pathways. A class of antibodies that bind to the specific HER3-forming epitope is described in Table 1. In one embodiment, the antibodies inhibit both the HER3 ligand-dependent and ligand-independent signaling pathways. In another embodiment, the antibodies bind to HER3 and do not block the binding of the HER ligand to the ligand binding site (that is, both the ligand and antibody can bind HER3 simultaneously).
[0246] [00246] The present invention provides antibodies that specifically bind to an HER3 protein (e.g., human HER3 and / or cinomologists), said antibodies comprise a VH domain having an amino acid sequence of SEQ ID NO: 15, 33, 51 , 69, 87, 105, 123, 141, 159, 177, 195, 213, 231,249, 267, 285, 303, 321,339, 357, and 375. The present invention provides antibodies that specifically bind to an HER3 protein (e.g. example, human HER3 and / or cinomologists), said antibodies comprise a VL domain having an amino acid sequence of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, 284, 302, 320, 338, 356, and 374. The present invention also provides antibodies that specifically bind to an HER3 protein (for example, human HER3 and / or cinomologists), said antibodies comprise a VH CDR having an amino acid sequence from any of the VH CDRs listed in Table 1, below. In particular, the present invention provides antibodies that specifically bind to an HER3 protein (for example, human HER 3 and / or cinomologists), said antibodies that comprise (or, in an alternative way, consisting of) CDRs one, two , three, four, five or more VH having an amino acid sequence from any of the VH CDRs listed in Table 1, below.
[0247] [00247] Other antibodies of the present invention include amino acids that have been mutated, yet at least 60, 70, 80, 90, 95, or 98 percent identity, in the CDR regions, with the CDR regions described in the sequences described in Table 1. In some modalities, which includes mutant amino acid sequences, in which no more than 1, 2, 3, 4 or 5 amino acids were mutated in the CDR regions, when compared to the CDR regions described in Table 1, the sequence described , while maintaining its specificity for epitopes of the original antibody
[0248] [00248] Other antibodies of the present invention include amino acids that have been mutated, yet at least 60, 70, 80, 90, 95, or 98 percent identity, in structural regions with the structural regions described in the sequences described in the Table 1. In some embodiments, which includes mutant amino acid sequences, in which no more than 1, 2, 3, 4, 5, 6, or 7 amino acids have been mutated in the structure regions, when compared to the structural regions represented in sequence described in Table 1, maintaining its specificity for the original antibody epitope. The present invention also provides nucleic acid sequences encoding VH, VL, the full-length heavy chain, and the full-length light chain of antibodies that specifically bind to an HER3 protein (e.g., human HER3 and / or cinomologists).
[0249] The HER3 antibodies of the present invention bind to the conformational epitope of HER3 which comprises the amino acid residues of domain 2 and domain 4 of HER3. Table 1: Examples of HER3 Antibodies of the Present Invention
[0250] [00250] Other antibodies of the present invention include those in which the amino acids and nucleic acids that code for the amino acids that have been mutated, but have at least 60, 70, 80, 90, 95, 96, 97, 98, and 99 percent identity with the sequences described in Table 1. In some embodiments, they include mutant amino acid sequences, in which no more than 1,2, 3, 4 or 5 amino acids were mutated in variable regions, when compared with the variable regions illustrated in the sequence described in Table 1, while retaining substantially the same therapeutic activity.
[0251] [00251] Since each of these antibodies or fragments thereof can bind to HER3, the full-length light chain, VH, VL, and the full-length heavy chain sequences (amino acid sequences and sequences nucleotide encoding amino acid sequences) can be "mixed and matched" to create other HER3 binding Antibodies of the present invention. Such "mixed and combined" HER3 binding antibodies can be tested using the binding assays known in the art (for example, ELISA and assays from others described in the Examples section). When these strands are mixed and matched, a VH sequence from a particular VH / VL pairing must be replaced by a structurally similar VH sequence. Likewise, a full-length heavy chain sequence from a given full-length heavy chain / full-length pairing light chain should be replaced by a structurally similar full-length heavy chain sequence. Likewise, a VL sequence from a particular VH / VL pair must be replaced by a structurally similar VL sequence. Likewise a full length of full length light chain sequence from a given full length heavy chain / pairing light chain should be replaced by a structurally similar full length light chain sequence. Thus, in one aspect, the present invention provides an isolated monoclonal antibody or a fragment thereof, having: a variable heavy chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 33, 51, 69, 87, 105, 123, 141, 159, 177, 195, 213, 231, 249, 267, 285, 303, 321, 339, 357, and 375, and a light chain variable region comprising a sequence of amino acids selected from the group consisting of SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, 284, 302, 320, 338 , 356, and 374; wherein the antibody specifically binds to HER3 (e.g., humans and / or cinomologists).
[0252] [00252] In another aspect, the present invention provides the HER3 binding antibodies that form the heavy chain and light chain, CDR1s CDR2s and CDR3 as described in Table 1, or combinations thereof. The VH amino acid sequences of CDR1s of the antibodies are shown in SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146 , 152, 164, 170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368. The VH amino acid sequences of CDR2s and antibodies are presented in SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129, 135, 147, 153, 165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255, 261, 273, 279, 291,297, 309, 315, 327, 333, 345, 351,363, and 369 The VH CDR3 antibody amino acid sequences are shown in SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82, 94, 100, 112, 118, 130, 136, 148, 154, 166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334, 346, 352, 364, and 370. The amino acid sequences of the CDR1s VL of the antibodies are shown in SEQ ID NOs: 5, 11.23, 29, 41.47, 59, 65, 77, 83, 95 , 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317 , 329, 335, 347, 353, 365, and 371. The VL amino acid sequences of the CDR2s of the antibodies are shown in SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348, 354, 366, and 372. The VL CDR3 antibody amino acid sequences are shown in SEQ ID NOs: 7, 13, 25, 31, 43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 133, 139, 151, 157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301,313, 319, 331,337, 349, 355, 367, and 373. CDR regions are outlined using the Kabat system (Kabat et al, (1991) Sequences of Proteins of Immunological Interesting, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91 to 3242 ,. Chothia et al, (. 1987) J. Mol. Biol 196: 901-917; Chothia et al, (1989) Nature 342: 877 to 883, and Al-Lazikani et al, (1997) J. Mol. Biol 273, 927 to 948).
[0253] [00253] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 3; a CDR3 of SEQ ID NO: 4, a variable region of the CDR1 light chain SEQ ID NO: 5, a CDR2 of SEQ ID NO: 6, and a CDR3 of SEQ ID NO: 7.
[0254] [00254] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 20, a CDR2 of SEQ ID NO: 21, a CDR3 of SEQ ID NO: 22, a variable region of the CDR1 light chain SEQ ID NO: 23, a CDR2 of SEQ ID NO: 24, and a CDR3 of SEQ ID NO: 25.
[0255] [00255] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 38, a CDR2 of SEQ ID NO: 39, a CDR3 of SEQ ID NO: 40, a variable region of the CDR1 light chain SEQ ID NO: 41, a CDR2 of SEQ ID NO: 42, and a CDR3 of SEQ ID NO: 43.
[0256] [00256] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 56, a CDR2 of SEQ ID NO: 57, a CDR3 of SEQ ID NO: 58, a variable region of the CDR1 light chain SEQ ID NO: 59, a CDR2 of SEQ ID NO: 60, and a CDR3 of SEQ ID NO: 61.
[0257] [00257] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 74, a CDR2 of SEQ ID NO: 75; a CDR3 of SEQ ID NO: 76, a variable region of the CDR1 light chain SEQ ID NO: 77, a CDR2 of SEQ ID NO: 78, and a CDR3 of SEQ ID NO: 79.
[0258] [00258] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 of the variable region of SEQ ID NO: 92, a CDR2 of SEQ ID NO: 93; a CDR3 of SEQ ID NO: 94, a variable region of the CDR1 light chain SEQ ID NO: 95, a CDR2 of SEQ ID NO: 96, and a CDR3 of SEQ ID NO: 97.
[0259] [00259] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 110; a CDR2 of SEQ ID NO: 111; a CDR3 of SEQ ID NO: 112, a variable region of the CDR1 light chain SEQ ID NO: 113; a CDR2 of SEQ ID NO: 114, and a CDR3 of SEQ ID NO: 115.
[0260] [00260] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 128; a CDR2 of SEQ ID NO: 129; a CDR3 of SEQ ID NO: 130, a variable region of the CDR1 light chain SEQ ID NO: 131; a CDR2 of SEQ ID NO: 132, and a CDR3 of SEQ ID NO: 133.
[0261] [00261] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 146; a CDR2 of SEQ ID NO: 147; a CDR3 of SEQ ID NO: 148; a CDR1 of the light chain variable region of SEQ ID NO: 149; a CDR2 of SEQ ID NO: 150, and a CDR3 of SEQ ID NO: 151.
[0262] [00262] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 164; a CDR2 of SEQ ID NO: 165; a CDR3 of SEQ ID NO: 166, a variable region of the CDR1 light chain SEQ ID NO: 167; a CDR2 of SEQ ID NO: 168, and a CDR3 of SEQ ID NO: 169.
[0263] [00263] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 182; a CDR2 of SEQ ID NO: 183; a CDR3 of SEQ ID NO: 184, a variable region of the CDR1 light chain SEQ ID NO: 185; a CDR2 of SEQ ID NO: 186, and a CDR3 of SEQ ID NO: 187.
[0264] [00264] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 200; a CDR2 of SEQ ID NO: 201; a CDR3 of SEQ ID NO: 202, a variable region of the CDR1 light chain SEQ ID NO: 203; a CDR2 of SEQ ID NO: 204, and a CDR3 of SEQ ID NO: 205.
[0265] [00265] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 218; a CDR2 of SEQ ID NO: 219; a CDR3 of SEQ ID NO: 220, a variable region of the CDR1 light chain SEQ ID NO: 221; a CDR2 of SEQ ID NO: 222, and a CDR3 of SEQ ID NO: 223.
[0266] [00266] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 236; a CDR2 of SEQ ID NO: 237; a CDR3 of SEQ ID NO: 238, a variable region of the CDR1 light chain SEQ ID NO: 239; a CDR2 of SEQ ID NO: 240, and a CDR3 of SEQ ID NO: 241.
[0267] [00267] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 254; a CDR2 of SEQ ID NO: 255; a CDR3 of SEQ ID NO: 256, a variable region of the CDR1 light chain SEQ ID NO: 257; a CDR2 of SEQ ID NO: 258, and a CDR3 of SEQ ID NO: 259.
[0268] [00268] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 272; a CDR2 of SEQ ID NO: 273; a CDR3 of SEQ ID NO: 274, a variable region of the CDR1 light chain SEQ ID NO: 275; a CDR2 of SEQ ID NO: 276, and a CDR3 of SEQ ID NO: 277.
[0269] [00269] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 290; a CDR2 of SEQ ID NO: 291; a CDR3 of SEQ ID NO: 292, a variable region of the CDR1 light chain SEQ ID NO: 293; a CDR2 of SEQ ID NO: 294, and a CDR3 of SEQ ID NO: 295.
[0270] [00270] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 308; a CDR2 of SEQ ID NO: 309; a CDR3 of SEQ ID NO: 310, a variable region of the CDR1 light chain SEQ ID NO: 311; a CDR2 of SEQ ID NO: 312, and a CDR3 of SEQ ID NO: 313.
[0271] [00271] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 326; a CDR2 of SEQ ID NO: 327; a CDR3 of SEQ ID NO: 328, a variable region of the CDR1 light chain SEQ ID NO: 329; a CDR2 of SEQ ID NO: 330, and a CDR3 of SEQ ID NO: 331.
[0272] [00272] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 344; a CDR2 of SEQ ID NO: 345; a CDR3 of SEQ ID NO: 346, a variable region of the CDR1 light chain SEQ ID NO: 347; a CDR2 of SEQ ID NO: 348, and a CDR3 of SEQ ID NO: 349.
[0273] [00273] In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain of CDR1 from the variable region of SEQ ID NO: 362; a CDR2 of SEQ ID NO: 363; a CDR3 of SEQ ID NO: 364; a CDR1 of the variable region of the light chain of SEQ ID NO: 365; a CDR2 of SEQ ID NO: 366, and a CDR3 of SEQ ID NO: 367.
[0274] [00274] In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO. 15 and VL of SEQ ID NO: 14. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 33 and VL of SEQ ID NO: 32. In a specific embodiment, an antibody that binds HER3 comprises a VH of SEQ ID NO: 51 and VL of SEQ ID NO: 50. In a specific embodiment, an antibody that binds to HER3 comprises a SEQ ID NO: 69 and VL of SEQ ID NO: 68. In a a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 87 and a VL of SEQ ID NO: 86. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 105 and VL of SEQ ID NO: 104. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 123 and VL of SEQ ID NO: 122. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 141 and VL of SEQ ID NO: 140. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 159 and VL of SEQ ID NO: 158. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 177 and VL of SEQ ID NO: 176. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 195 and VL of SEQ ID NO: 194. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 213 and VL of SEQ ID NO: 212. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 231 and VL of SEQ ID NO: 230. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 249 and VL of SEQ ID NO: 248. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 267 and VL of SEQ ID NO: 266. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 285 and VL of SEQ ID NO: 284. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 303 and VL of SEQ ID NO: 302. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 321 and a VL of SEQ ID NO: 320. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 339 and VL of SEQ ID NO: 338. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 357 and VL of SEQ ID NO: 356. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 375 and VL of SEQ ID NO: 374.In modality one, HER3 Antibodies are anetiquetaonist antibodies. In certain embodiments, an antibody that binds to HER3 is an antibody that is described in Table 1.
[0275] [00275] As used in the present invention, a human antibody comprises the heavy or light chain variable regions or full or light length heavy chains, which are "the product of" or "derived from" a particular germline sequence if the variable regions of full-length chains or the antibody are obtained from a system that uses the germline human immunoglobulin genes. Such systems include the immunization of a transgenic mouse carrying human immunoglobulin genes, with the antigen of interest, or the search for a library of human immunoglobulin genes presented on phages with the antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of the human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greater% identity) to the human antibody sequence. A human antibody that is "the product of" or "derived from" an immunoglobulin sequence of the particular human germline may contain differences in amino acids, compared to the sequence of the germline, due to, for example, somatic mutations that occur in a natural way or intentionally introducing local-directed mutations. However, the VH or VL framework regions of a selected human antibody are typically at least 90% identical in the amino acid sequence of an amino acid sequence encoded by a human germline immunoglobulin gene and contain the amino acid residues that identify the human antibody as being human when compared to those of the immunoglobulin germline amino acid sequences from other species (for example, the murine germline sequences). In certain cases, a human antibody can be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in the amino acid sequence for the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a recombinant human antibody will exhibit more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene in the VH or VL framework regions. In certain cases, the human antibody may exhibit more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene. The different versions of the germ line using the VH and VL of the germ line of sequences of a representative number of HER3 Antibodies are shown in Table 2, using Kabat. CDR positions are highlighted in bold. The notation used in the Tables with the germline sequences is as follows: -MOR10701 VH_3-07 means MOR10701 the CDR loops in regions of VH structure of the germline sequence from 3 to 07 (nomenclature is in accordance with Vbase), MOR10703 -VK_L1 means CDR of MOR10703 in the germ line of the VK_L1 structure regions, where VK is the kappa light chain. Table 2: Different versions of the germline of a selected number of representative antibodies
[0276] [00276] Any combination in the VH germline SEQuences with the JH segments can be used. Representative examples of combinations are shown in Table 5. Table 5: Representative examples of combinations in the SEQuences of germline VH with a JH segment.
[0277] [00277] Any combination in the SEQuences of germline VL with a JK segment can be used. Representative examples of combinations are shown in Table 6. Table 6: Representative examples of combinations in the SEK VK germline sequences with a JK segment
[0278] [00278] Once VH has been combined with JH and VK with JK, then any combination of VH or JH with VK or JK, can be used. In one embodiment, any of the VH germline regions can be combined with any of the VK germline (VL) regions for each antibody. A representative number of examples of combinations are shown in Table 7. Table 7: Representative examples of combinations of germline sequences
[0279] [00279] In one embodiment, the present invention relates to a variable region of heavy chain comprising a sequence of Xaa1-Xaa2 - HCDR1 HCDR2 HCDR3-Xaa3-Xaa4-wherein the heavy chain HCDR1, HCDR2, HCDR3 are any CDRs of heavy chain selected from Tables 1 and 2. For illustrative purposes only, the sequence can be:
[0280] [00280] Xaa1 - SYAMS - Xaa2 - AINSQGKSTYYADSVKG - Xaa3 -WGDEGFDI - Xaa4 (SEQ ID NO: 493), where,
[0281] [00281] Xaa1 is a structure region of any 30 amino acids;
[0282] [00282] Xaa2 is any region of structure of 14 amino acids;
[0283] [00283] Xaa3 is a 32-amino acid structure region;
[0284] [00284] Xaa4 is a structure region of any 11 amino acids;
[0285] [00285] In one embodiment, the present invention relates to a variable region of light chain comprising a sequence of Xaa1-LCDR1 LCDR2-Xaa2-Xaa3-Xaa4 - LCDR3, wherein the LCDR1, LCDR2, LCDR3 light chain are any Light chain CDRs selected from Tables 1 and 2. For illustrative purposes only, the sequence can be:
[0286] [00286] Xaa1 - RASQGISNWLA - Xaa2 - GASSLQS - Xaa3 -QQYSSFPTT - Xaa4 (SEQ ID NO: 494), where,
[0287] [00287] Xaa1 is a structure region of any 23 amino acids;
[0288] [00288] Xaa2 is a structure region of any 15 amino acids;
[0289] [00289] Xaa3 is a structure region of any 32 amino acids; and
[0290] [00290] Xaa4 is a structure region of any 10 amino acids.
[0291] [00291] The antibodies described in the present invention can be derived from single chain antibodies, diabodies, domain antibodies, nanobodies, and unicibodies. A "single chain antibody" (scFv) consists of a single polypeptide chain comprising a VL domain linked to a VH domain, where the VL domain and the VH domain are paired to form a monovalent molecule. The single chain antibody can be prepared according to the method known in the art (see, for example, Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad I know, US 85: 5879 to 5883). A "disbud" consists of two chains, each chain comprising a variable region of the heavy chain linked to a variable region of the light chain on the same polypeptide chain linked by means of a short peptide linker, where the two regions on the same chain do not match with each other but with the complementary domains of another chain, in order to form a bispecific molecule. Methods of preparing bodies are known in the art (See, for example, Holliger et al., (1993) Proc. Natl. Acad. Sci. US 90: 6444 to 6448, and Poljak et al., (1994) Structure 2: 1121 to 1123). Domain antibodies (DABS) are small functional antibody-binding units, which correspond to the variable regions of both heavy or light chains of antibodies. Domain antibodies are well expressed in yeast, bacteria, and mammalian cell systems. Additional details of domain antibodies and methods of producing them are known in the art (see, for example, US Patent Nos. 6,291,158;. 6582915; 6593081; 6172197; 6696245; European Patents 0 368,684 and 0.616,640; WO05 / 035572, WO04 / 101790, nanobodies WO04 / 081026, WO04 / 058821, WO04 / 003019 and WO03 / 002609. are derived from the heavy chains of an antibody A nanobody typically comprises a single variable domain and two constant domains (CH2 and CH3) and retains the antigen-binding capacity of the original antibody to nanobodies can be prepared by methods known in the art (See, for example, US Patent No. 6,765,087, US Patent No. 6,838,254, WO 06/079372) Unicibodies consist of a light chain and a heavy chain of an IgG4 antibody.Unibodies can be made by removing the hinge region of IgG4 antibodies.More details of the antibodies and methods for their preparation can be found in WO2007 / 059782. Homologous Antibodies
[0292] [00292] In yet another embodiment, the present invention provides an antibody or a fragment thereof comprising amino acid sequences that are homologous to the sequences described in Table 1, and said antibody binds to an HER3 protein (for example, HER3 and / or cinomologists), and retains the desired functional properties of these antibodies described in Table 1.
[0293] [00293] For example, the present invention provides an isolated monoclonal antibody (or a fragment of the same functional), which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence amino acid that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 33, 51, 69, 87, 105, 123, 141, 159, 177, 195, 213, 231, 249, 267, 285, 303, 321, 339, 357, and 375, the variable region of the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, 194, 212, 230, 248, 266, 284, 302, 320, 338, 356, and 374, the antibody binds to HER3 (for example, human HER3 and / or cinomologists) and neutralizes signaling activity HER3, which can be measured in a phosphorylation assay or other HER signaling measure (for example, HER3 phospho-assays, Akt phospho-assays, cell proliferation, and ligand block assays as described in the Examples ). Also included within the scope of the present invention are the parental heavy and light chain variables in the nucleotide sequences and the full length sequences of the heavy and light chain optimized for expression in a mammalian cell. Other antibodies of the present invention include the amino acids or nucleic acids that have been mutated, yet at least 60, 70, 80, 90, 95, or 98 percent identity% to the sequences described above. In some embodiments, they include mutant amino acid sequences, in which no more than 1,2, 3, 4 or 5 amino acids were mutated through the deletion of amino acids, insertion or substitution of variable regions, when compared to the variable regions illustrated in the sequence described above.
[0294] [00294] In other modalities, the VH and / or VL amino acid sequences can be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set out in Table 1. In other embodiments, the VH and / or VL amino acid sequences can be identical except for an amino acid substitution in no more than 1,2,3,4 or 5 amino acid positions. An antibody that has high VH and VL regions (that is, 80% or more) for the identity of VH and VL regions of the antibodies described in Table 1 can be obtained through muetiquetaênese (for example, site-directed or mediated muetiquetaênese) PCR), followed by testing the encoded antibody altered to the retained function using the functional assays described in the present invention.
[0295] [00295] In other modalities, the variable regions in the SEQUENCES of light chain and / or heavy chain nucleotides can be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences defined above.
[0296] [00296] As used in the present invention, the "identity tag" between the two strings is a function of the number of identical positions shared by the strings (i.e.,% identity is equal to the number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The sequence comparison and the determination of the identity tag between the two sequences can be performed using a mathematical algorithm, as described in the following non-limiting examples.
[0297] [00297] Additionally or alternatively, the protein sequences of the present invention can still be used as a "search string" to perform a search on public databases to, for example, identify related sequences. For example, these searches can be performed using the BLAST program (version 2.0) by Altschul et al., (1990) J.Mol. Biol. 215: 403 to 10. Antibodies with Conservative Modifications
[0298] [00298] In certain embodiments, an antibody of the present invention has a heavy chain variable region comprising CDR1, CDR2 and CDR3 and the sequences of a light chain variable region comprising CDR1, CDR2 and CDR3, where one or more of these sequences of CDR has the specified amino acid sequences based on the antibodies described herein or the conservative modifications therein, and wherein the antibodies retain the desired functional properties of the HER3 binding antibodies of the present invention.
[0299] [00299] In this way, the present invention provides an isolated monoclonal antibody HER3, or a fragment thereof, consisting of a heavy chain variable region comprising CDR1, CDR2 and CDR3 and the sequences of a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, in which: the amino acid sequences of the heavy chain variable region CDR1 are selected from the group consisting of SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152, 164, 170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368, and their conservative modifications; the amino acid sequences of the variable region of the heavy chain of CDR2 are selected from the group consisting of SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111 , 117, 129, 135, 147, 153, 165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255, 261, 273, 279, 291, 297, 309, 315, 327, 333 , 345, 351, 363, and 369 and conservative modifications thereof; the amino acid sequences of the variable region of the CDR3 heavy chain are selected from the group consisting of SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82, 94, 100, 112 , 118, 130, 136, 148, 154, 166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334 , 346, 352, 364, and 370 and conservative modifications thereof; the amino acid sequences of the variable region of the CDR3 light chain are selected from the group consisting of SEQ ID NOs: 5, 11.23, 29, 41.47, 59, 65, 77, 83, 95, 101, 113 , 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317, 329, 335 , 347, 353, 365, and 371 and their conservative modifications; the amino acid sequences of the CDR2 light chain variable regions are selected from the group consisting of SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348, 354, 366, and 372, and their conservative modifications; the amino acid sequences of the CDR3 light chain variable regions are selected from the group consisting of SEQ ID NOs: 7, 13, 25, 31.43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 133, 139, 151, 157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301, 313, 319, 331, 337, 349, 355, 367, and 373, and their conservative modifications; the antibody or fragment thereof specifically binds to HER3, and neutralizes the activity of HER3 that inhibits an HER signaling pathway, which can be measured in a phosphorylation assay or can be measured by means of another HER signaling ( for example, HER3 phospho-assays, Akt phospho-assays, cell proliferation, and binding block assays as described in the Examples). Antibodies that bind to the same epitope
[0300] [00300] The present invention provides antibodies that interact with (for example, by preventing, steric binding, stabilizing / destabilizing spatial distribution) the same epitope as the HER3 binding antibodies described in Table 1 and in fig. 7. Additional antibodies can therefore be identified based on their ability to cross-compete (for example, with the purpose of competitively inhibiting binding in a statistically significant manner) with other antibodies of the present invention in binding assays. HER3. The ability of a test antibody to inhibit the binding of the antibodies of the present invention to an HER3 protein (for example, human HER3 and / or cinomologists) demonstrates that the test antibody can compete with the HER3 binding antibody, an antibody such as it can, according to non-limiting theory, bind to the same epitope or to one (for example, a similar or spatially proximal structure) related to the HER3 protein as the antibody with which it competes. In a certain embodiment, the antibody that binds to the same epitope in HER3 as the antibodies of the present invention is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in this document.
[0301] [00301] In one embodiment, the antibody or its fragments bind to both domain 2 and domain 4 of HER3 to hold HER3 in an inactive conformation that avoids exposing a circuit present within the dimerization domain 2 This prevents heterodimerization with other family members, such as HER1, HER2 and HER4. Fragment antibodies inhibit both ligand-dependent and ligand-independent HER3 signal transduction.
[0302] [00302] In another embodiment, the antibody or a fragment of it binds to both domain 2 and domain 4 of HER3 and without blocking the simultaneous binding of a ligand such as the neuregulin HER3. Although it is not necessary to provide a theory, it is possible that the antibody or a fragment of it binds to domain 2 and domain 4 of HER3, HER3 holds in an inactive conformation, without blocking the ligand binding site in HER3. In this way, an HER3 ligand (for example, neuregulin) is able to bind to HER3, at the same time as the antibody or its fragment.
[0303] [00303] The antibodies of the present invention, or fragments thereof, inhibit both activation of the dependent and independent of the HER3 ligand, without preventing binding of the ligand. This is considered advantageous for the following reasons:
[0304] [00304] (I) The therapeutic antibody will have clinical utility for a broader spectrum of tumors than an antibody that targets a single HER3 activation mechanism (i.e., ligand-independent or ligand-dependent), since different types of tumors are triggered by each mechanism.
[0305] [00305] (Ii) The therapeutic antibody would be effective in tumor types in which both HER3 activation mechanisms are simultaneously involved. An antibody targeting a single HER3 activation mechanism (ie, ligand-dependent or ligand-independent) that exhibits little or no efficacy in these types of tumors
[0306] [00306] (Iii) The effectiveness of an antibody that inhibits ligand-dependent activation of HER3 without preventing ligand binding would be less likely to be adversely affected by increasing ligand concentrations. Such a procedure would imply both efficacy in a type of tumor driven by very high concentrations of HER3 ligand and a sanction of reduced resistance to drugs, where resistance is mediated through the over-regulation of HER3 ligands.
[0307] [00307] (Iv) an antibody that inhibits activation by HER3 by stabilizing the inactive form would be less prone to resistance to drugs triggered through the alternative mechanisms of HER3 activation.
[0308] Consequently, the antibodies of the present invention can be used to treat conditions where existing therapeutic antibodies are clinically ineffective. Engineering and Modified Antibodies
[0309] [00309] An antibody of the present invention can be prepared using an antibody that has one or more of the VH and / or VL sequences shown in the present invention as a starting material for generating a modified antibody, which the modified antibody may have altered the starting antibody properties. An antibody can be manipulated by modifying one or more residues within one or both of the variable regions (i.e., VH and / or VL), for example, within one or more CDR regions and / or within a or more structure regions. Additionally or alternatively, an antibody can be manipulated by modifying residues within the constant region (s), for example, to alter the antibody's effective function (s) .
[0310] [00310] One type of variable region engineering that can be performed is CDR grafting. The antibodies interact with target antigens predominantly through amino acid residues that are located in the complementarity of the six heavy and light determinant regions (CDR). For this reason, amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific antibodies that mimic the properties of antibodies that naturally occur by constructing expression vectors that include the sequences of CDR of the naturally occurring specific antibody grafted into the structural sequences of a different antibody with different properties (see, for example, Riechmann et al, (1998) Nature 332: 323 to 327; Jones et al, (1986) Nature 321: 522 to 525; Queen et al, (1989) Proc Natl. Acad, US 86: 10029 to 10033, US Patent No. 5,225,539 to Winter, and US Patent No. 5,530,101; 5,585,089; 5,693 .762 and 6,180,370 by Queen et al).
[0311] Accordingly, another embodiment of the present invention relates to an isolated HER3 binding monoclonal antibody, or fragment thereof, comprising the sequences of a heavy chain variable region CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152, 164, 170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129, 135 , 147, 153, 165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255, 261, 273, 279, 291, 297, 309, 315, 327, 333, 345, 351, 363 , and 369; CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82, 94, 100, 112, 118, 130, 136 , 148, 154, 166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334, 346, 352, 364 , and 370, respectively, and a light chain variable region having the CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, 23, 29, 41.47, 59, 65, 77, 83, 95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317, 329, 335, 347, 353, 365, and 371; CDR 2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138 , 150, 156, 168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348, 354, 366 , and 372, and CDR3 sequences consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 13, 25, 31.43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 135, 139, 151, 157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301, 313, 319, 331, 337, 349, 355, 367, and 373, respectively. Thus, antibodies that contain the CDR VH and VL sequences of monoclonal antibodies, but which may contain structural sequences different from those antibody structural sequences. They can be obtained from public DNA databases or published references that include the germline antibody gene sequences. For example, human germline DNA sequences for variable and heavy chain genes from the variable region can be found in the "vessel" of the human germline sequence database (available on the Internet at www.mrc-cpe.cam .ac.uk / Vbase), as well as in Kabat et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91 to 3242 ,. Chothia et al, (1987) J. Mol. Biol .. Biol. 196: 901 to 917; Chothia et al, (1989) Nature 342: 877 to 883; . And Al-Lazikani et al, (1997) J. Mol. Biol .. Biol. 273: 927 to 948; Tomlinson et al, (1992) J. fol .. Biol. 227: 776 to 98, and Cox et al, (1994) Eur .. J Immunol. 24: 827 to 836; the contents of each of which are in the present invention expressly incorporated by reference.
[0312] [00312] An example of structural sequences for use in the antibodies of the present invention are those that are structurally similar to those in the Structural SEQUENCES used by antibodies selected from the present invention, for example, consensus sequences and / or structural sequences used by the monoclonal antibodies of the present invention. The VH CDR1, 2 and 3, as well as the VL CDR1, 2 and 3 sequences, can be grafted into structural regions that have the sequence identical to that found in the germline immunoglobulin gene from which the frame sequence is derived, or in the CDR sequences, they can be grafted into structural regions that contain one or more mutations compared to the germline sequences. For example, it has been found that, in certain cases, it is beneficial to mutate residues in the framework regions to maintain or improve the antigen-binding capacity of the antibody (see, for example, US Patent No. 5,530,101; 5,585,089 ; 5,693,762 and 6,180,370 by Queen et al).
[0313] [00313] Another type of variable region modification is the mutation of amino acid residues within the VH and / or VL regions of CDR1, CDR2 and / or CDR3 in order to improve one or more binding properties (for example, affinity) of the antibody of interest, known as "maturation affinity". Site-directed muetiquetahnesis or PCR-mediated muetiquetahnesis can be performed to introduce the mutation (s) and the effect on antibody binding, or other functional property of interest, can be evaluated in vitro or in in vivo assays , as described in the present invention and provided in the Examples. Conservative modifications (as discussed above) can be introduced. Mutations can be substitutions, additions or deletions of amino acids. In addition, normally no more than one, two, three, four or five residues from a CDR region are altered.
[0314] Therefore, in another embodiment, the present invention provides isolated HER3 monoclonal binding antibodies, or fragments thereof, consisting of a variable region of the heavy chain having: a VH CDR1 region consisting of an amino acid sequence selected from the group that has SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134,146, 152, 164, 170, 182 , 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368, or a sequence of amino acids that has one, two, three, four or five amino acid substitutions, deletions or additions, compared to SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152, 164, 170, 182, 188, 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368, a VH CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129, 135, 147, 153, 165, 171, 183, 189, 201, 207 , 219, 225, 237, 243, 255, 261,273, 279, 291,297, 309, 315, 327, 333, 345, 351,363, and 369, or an amino acid sequence having one, two, three, four or five substitutions, deletions or amino acid additions, compared to SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129, 135, 147, 153, 165 , 171, 183, 189, 201,207, 219, 225, 237, 243, 255, 261, 273, 279, 291, 297, 309, 315, 327, 333, 345, 351,363, and 369, a VH CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82, 94, 100, 112, 118, 130, 136, 148, 154, 166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334, 346, 352, 364, and 370 , or a sequence of amino acids that has one, two, three, four or five amino acid substitutions, deletions or additions in relation to SEQ ID NO: 4, 10, 22, 28, 40, 46, 58, 64, 76, 82, 94, 100, 112, 118, 130, 136, 148, 154, 166, 172, 184 , 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334, 346, 352, 364, and 370, a CDR1 VL region having a amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11.23, 29, 41.47, 59, 65, 77, 83, 95, 101, 113, 119, 131, 137, 149, 155 , 167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311, 317, 329, 335, 347, 353, 365, and 371, or an amino acid sequence that has one, two, three, four or five amino acid substitutions, deletions or additions, compared to SEQ ID NOs: 5, 11, 23, 29, 41, 47, 59, 65, 77 , 83, 95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299 , 311,317, 329, 335, 347, 353, 365, and 371, a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348, 354, 366, and 372, or a sequence of amino acids that has one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 6, 12, 24, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120 , 132, 138, 150, 156, 168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348 , 354, 366, and 372, and a VR CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 13, 25, 31.43, 49, 61.67, 79, 85 , 97, 103, 115, 121, 135, 139, 139, 151, 157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301 , 313, 319, 331, 337, 349, 355, 367, and 373, or an amino acid sequence that has one, two, three, four or five substitutions for it amino acid additions or additions in relation to SEQ ID NO: 7, 13, 25, 31, 43, 49, 61, 67, 79, 85, 97, 103, 115, 121, 135, 139, 139, 151, 157 , 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301, 313, 319, 331,337, 349, 355, 367, and 373. Grafting antibody fragments onto alternative structures or scaffolding
[0315] [00315] A wide variety of antibody / immunoglobulin structures or scaffolds can be used, as long as the resulting polypeptide includes at least one binding region that specifically binds HER3. Such structures or scaffolding include the 5 main human immunoglobulin idiotypes, or fragments thereof, and include immunoglobulins from other animal species, preferably with humanized aspects. The new structures, scaffolding and fragments continue to be discovered and developed by those skilled in the art.
[0316] [00316] In one aspect, the present invention relates to the generation of antibodies based on the non-immunoglobulin using non-immunoglobulin scaffolds in which CDRs of the present invention can be grafted. Known or future non-immunoglobulin structures and scaffolds can be employed, as long as they comprise a specific binding region for the target protein HER3 (for example, human HER3 and / or cinomologists). Known non-immunoglobulin structures or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-drugs (Trubion Pharmaceuticals Inc., Seattle, WA), (Avidia maxybodies, Inc., Mountain View, CA ), protein A (Afficorpo AG, Sweden), and affilin (gamma-crystalline or ubiquitin) (Scil Proteínas GmbH, Halle, Germany).
[0317] [00317] The supports are based on type III domain fibronectin fibronectin (e.g., the tenth module of type III fibronectin (10 FN3 domains)). Type III domain fibronectin has 7 or 8 beta chains that are distributed between two beta sheets, which they themselves pack against each other for the purpose of forming the protein nucleus, and other loops containing (analogous to CDRs) that bind beta chains to each other and are exposed solvents. There are at least three loops at each end of the beta sandwich sheet, where the border is the boundary of the protein perpendicular to the direction of the beta chains (see U.S. 6,818,418). These fibronectin-based supports are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire IgG camel and llama antigen recognition unit. Because of this structure, the non-immunoglobulin antibody mimics antigen-binding properties that are similar in nature and affinity to antibodies. These supports can be used in a loop randomization and in vitro shuffling strategy that is similar to the antibody affinity maturation process in vivo. These fibronectin-based molecules can be used as supports where the loop regions of the molecule can be replaced with CDR of the present invention, using conventional cloning techniques.
[0318] [00318] Ankyrine technology is based on the use of proteins with ankyrin repetition modules derived as scaffolding to support the variable regions that can be used to bind to different targets. The ankyrin repeat module is a 33-amino acid polypeptide consisting of two anti-parallel α-helices and a β-turn. The binding of the variable regions is mainly optimized using the ribosome display.
[0319] [00319] Avimers are derived from the natural A domain containing protein such as HER3. These domains are used by nature for protein-protein interactions and in humans with more than 250 proteins they are structurally based on A domains. Avimers consist of a number of different "domain A" monomers (2-10) linked through elements of amino acid binding. Avimers can be created, which can bind to the target antigen using the methodology described in, for example, U.S. Patent Application No. Publication No. 20040175756; 20050053973; 20050048512 and 20060008844.
[0320] [00320] Afficorpo affinity ligands are simple, small proteins composed of a bundle of three helices based on the scaffolding of one of the IgG binding domains of protein A. Protein A is a surface protein of the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, of which 13 are chosen at random to generate afficorpo libraries with a large number of linker variants (See, for example, U.S. 5,831,012). Afficorpo antibody molecules mimic, have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. Despite their small size, the binding site of the afficorpo molecules is similar to that of an antibody.
[0321] [00321] Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocalins, a broad group of small, solid proteins, which are normally involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Various natural lipocalins occur in human tissues or in body fluids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops at the top of a rigid structure. However, in contrast to antibodies or fragments of the same recombinants, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being only slightly larger than a single immunoglobulin domain. The set of four loops, which makes the connection pouch, has pronounced structural plasticity and tolerates a variety of side chains. The binding site can, in this way, be remodeled in a specific way to recognize prescribed target molecules in a different way, with high affinity and specificity. A protein of the lipocalin family, the bilin-binding protein (BBP) of Brassicae Pieris has been used to develop anticalins by muetiquetaênese of the set of four loops. An example of a patent application that describes anticalins is in PCT Publication No.WO 199916873.
[0322] [00322] Affiline molecules are small non-immunoglobulin proteins that are designed for specific affinities for proteins and small molecules. the new affiline molecules can be quickly selected from two libraries, each of which is based on a different human protein derived from scaffolding. The affiline molecules do not show any structural homology to the immunoglobulin proteins. Currently, two affilina supports are employed, one of which is gamma crystalline, a structural protein of the human eye lens and the other is the proteins of the "ubiquitin" superfamily. Both human supports are very small, show stability at high temperatures and are almost resistant to changes in pH and denaturing agents. This high stability is mainly due to the expanded beta-sheet structure of the proteins. Examples of proteins derived from crystalline gamma are described in WO200104144 and examples of "ubiquitin" type proteins are described in WO2004106368.
[0323] [00323] Epitope protein (PEM) mimetics are medium-sized, cyclic peptides, similar molecules (MW 12 kDa) that mimic secondary beta-hairpin structures of proteins, the primary secondary structure involved in protein-protein interactions.
[0324] [00324] In some modalities, the Fabs are converted to the silent IgG1 format, changing the Fc region. For example, the antibodies in Table 1 can be converted to the IgG format. Human or humanized antibodies
[0325] [00325] The present invention provides fully human antibodies that specifically bind to an HER3 protein (for example, humans and / or cinomologists / mouse / HER3 mouse). In comparison to chimeric or humanized antibodies, the human HER3-binding antibodies of the present invention further reduced antigenicity when administered to humans.
[0326] [00326] Human HER3 binding antibodies can be generated using methods that are known in the art. For example, the technology used for converting humanization of non-human antibodies to human antibody engineering. US Patent Publication No. 20050008625 describes an in vivo method for replacing an antibody non-human variable region with an antibody human variable region, maintaining it or providing better binding characteristics over that of the non-human antibody . The method is based on the guided replacement of the epitope of variable regions of a non-human reference antibody with a fully human antibody. The resulting human antibody is generally structurally related to the reference non-human antibody, but it binds to the same epitope on the same antigen as the reference antibody. Briefly, the series of guided epitope complementarity substitution approach is activated by creating a competition in cells between a "competitor" and a library of different hybrids of the reference antibody ("test antibodies") for binding to limit the amounts of antigen in the presence of a reporter system that responds to the binding of the antibody to the test antigen. The competitor may be the reference antibody, or a derivative thereof, as a single chain Fv fragment. The competitor may also be a natural or artificial antigen ligand that binds to the same epitope as the reference antibody. The only requirements are those of the competitor that binds to the same epitope as the reference antibody, and that competes with the reference antibody for binding to the antigen. The test antibodies have a binding to the V region antigen in common from the non-human reference antibody, and the other V region randomly selected from a diverse source, such as a human antibody repertoire library. The common V region of the reference antibody serves as a guide, the placement of the test antibodies in the same epitope on the antigen, and in the same orientation, so that the selection is tilted for the highest antigen-binding fidelity to the reference antibody .
[0327] [00327] Many types of reporter systems can be used to detect interactions between desired test and antigen antibodies. For example, the reporter complement fragments can be linked to test antigen and antibody, respectively, so that activation of the reporter by fragment complementation only occurs when the test antibody binds to the antigen. When the antibody-antigen and reporter-fragment fusion tester is co-expressed with a competitor, reporter activation becomes dependent on the test antibody's ability to compete with the competitor, which is proportional to the antibody's affinity for the test antigen. Other reporter systems, which may be used include the reactivator of a reporter reactivation self-inhibiting system (RAIR) as described in US Patent Application No. Ser. 10/208, 730 (Publication No. 20030198971), or of the competitive activation system described in US Patent Application No. Ser. 10/076, 845 (Publication No. 20030157579).
[0328] [00328] With the epitope complementarity guided substitution system series, the selection is made to identify the cells expressing a single test antibody along with the competitor, antigen and reporter components. In these cells, each test antibody competes one-on-one with the competitor for binding to a limiting amount of antigen. Reporter activity is proportional to the amount of antigen bound to the test antibody, which in turn is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. The antibodies used are initially selected based on their activity in relation to that of the reference antibody, when expressed as the test antibody. The result of the first round of selection is a set of antibody "hybrids", each of which consists of the same non-human V region of the reference antibody and a human V region from the library, and each of which binds to the same epitope on the antigen as the reference antibody. One or more of the hybrid antibodies selected in the first round will have an affinity for the antigen that is comparable to or greater than that of the reference antibody.
[0329] [00329] In the second stage of substitution of the V region, the human V regions selected in the first stage are used as a guide for the selection of human substitutions for the remaining reference non-human antibody V region with a diverse library of human V regions. cognates. The hybrid antibodies selected in the first round can also be used as competitors for the second round of selection. The result of the second round of selection is a set of completely human antibodies that differ structurally from the reference antibody, but which compete with the reference antibody for binding to the same antigen. Some of the selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Among these selected human antibodies, one or more binds to the same epitope with an affinity that is comparable to or greater than that of the reference antibody.
[0330] [00330] Using one of the mouse or chimeric HER3 binding antibodies described above as the reference antibody, this method can be easily used to generate human antibodies that bind to human HER3 with the same binding specificity and binding affinity or best. In addition, such human antibody-binding HER3 can also be obtained commercially from companies that habitually produce human antibodies, for example, KaloBios, Inc. (Mountain View, CA). Camelid Antibodies
[0331] [00331] Antibody proteins obtained from members of the camel and dromedary family (Camelus bactrianus and Calelus dromaderius) including members of the new world, such as llama species (Lama paccos, Dalai Lama and glama vicugna) were characterized in terms of size, complexity structural and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature have light chains, and are thus structurally distinct from the structure of the typical four quaternary chain having two heavy chains and two light chains of antibodies, from other animals . See PCT / EP93 / 02214 (WO 94/04678 published on March 3, 1994).
[0332] [00332] The region of the camelid antibody that is the only small variable domain identified as VHH can be obtained through genetic engineering in order to produce a small protein with a high affinity for the target, which results in a low molecular weight of the protein-derived antibody known as a "camelid nanobody". See U.S. Patent Number 5,759,808 issued June 2, 1998, see also Stijlemans et al, (2004) J Biol Chem 279: 1256 to 1261; . Dumoulin et al, (2003) Nature 424: 783 to 788, Pleschberger et al, (2003) Bioconjugate Chem 14: 440-448; Cortez-Retamozo et al, (2002) Int J Cancer 89: 456 to 62, and Lauwereys et al, (1998) EMBO J 17: 3512 to 3520. Engineered antibody libraries and fragments of camelid antibodies are commercially available, for example , from Ablynx, Ghent, Belgium. (For example, US20060115470 ,. Domantis (US20070065440, US20090148434) As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles the human sequence , that is, the Nanocorpo can be “humanized.” In this way, the low natural antigenicity of camelid antibodies to humans can be further reduced.
[0333] [00333] The camelid nanocorp has a molecular weight of approximately one tenth of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. A consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, that is, camelid nanobodies are useful as antigen-detecting reagents, which are otherwise cryptic using classic immunological techniques, and as possible therapeutic agents. In this way, yet another consequence of the small size is that a camelid nanocorp can inhibit, as a result of binding to a specific location, with a narrow groove or slit of a target protein, and therefore can serve in a ability that more closely resembles the function of a classic low molecular weight drug than that of a classic antibody.
[0334] [00334] The low molecular weight and the compact size result even more in camelid nanobodies being extremely thermostable, stable at extreme pH and proteolytic digestion, and poorly antigenic. Another consequence is that the camelid nanobodies easily move from the circulatory system to the tissues, and even cross the blood-brain barrier, and can treat disorders that affect the tissue of the nervous system. Nanobodies can further facilitate drug transport across the blood-brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These characteristics combined with low antigenicity for humans indicate great therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells, such as E. coli, and are expressed as bacteriophage fusion proteins and are functional.
[0335] [00335] Accordingly, a feature of the present invention is an antibody or decamelid Nanocorp having high affinity for HER3. In certain embodiments of the present invention, the antibody or camelid nanocorp is produced naturally in the camelid animal, that is, it is produced by immunizing the next camelid with HER3 or a fragment of the same peptide, using techniques described in the present invention for others antibodies. Alternatively, the HER3-binding camelid nanocorp was designed, that is, produced by selecting, for example, from a phage library appropriately displaying the multi-tasked protein camelid nanobodies using panning procedures with HER3 as a target, as described in the examples in the present invention. The manipulated nanobodies can be further customized by means of genetic engineering to have a half-life of a recipient individual from 45 minutes to two weeks. In a specific embodiment, the antibody or camelid Nanocorp is obtained by CDR grafting the heavy or light chain sequences of the human antibodies of the present invention into Nanocorp or single antibody domain sequences of the framework, as for example described in PCT / EP93 / 02214. In one embodiment, the antibody or camelid nanocorp binds to at least one of the following residues: HER3 Asn266, Lys267, Thr269, Leu268, Gln271, Glu273, Pro274, Pro276, Asn275, His277, Asn315, Asp571, Pro583, His584, Ala596, Lys597. In one embodiment, the decamelid antibody or Nanocorp binds to at least one of the following residues: HER3 Tyr265, Lys267, Leu268, Phe270, Gly582, Pro583, Lys597, Lys602, Ile600, Arg611, Glu609, Pro612, Cys613, His614, Glu615. Bispecific molecules and multivalent antibodies
[0336] [00336] In another aspect, the present invention features bispecific or multispecific biparatopic molecules comprising an HER3 binding antibody, or a fragment thereof, of the present invention. An antibody of the present invention, or fragments thereof, can be derivatized or linked to another functional molecule, for example, another peptide or protein (for example, another antibody or a linker for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the present invention can, in fact, be derivatized or linked to more than one other functional molecule to generate biparatopic or multi-specific molecules that bind to more than two different types of binding sites and / or target molecules ; such biparatopic or multi-specific molecules. To create a bispecific molecule of the present invention, an antibody of the present invention can be functionally linked (for example, by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other binding molecules, such as another antibody , antibody fragment, the binding peptide or mimetic, such that a bispecific molecule results.
[0337] [00337] Other clinical benefits can be provided by binding two or more antigens from within an antibody (Coloma et al, (1997) ,. Merchant et al, (1998), Alt et al, (1999), Zuo et al, (2000); Lu et al, (2004) ,. Lu et al, (2005), Marvin et al, (2005), Marvin et al, (2006), Shen et al, (2007), Wu et al , (2007), DiMasi et al, (2009), Michaelson et al, (2009)). (Morrison et al, (1997) Nature Biotech 15: 159 to 163, Alt et al Letters (1999) FEBS 454: 90 to 94; Zuo et al, (2000) Protein Engineering 13: 361 to 367 ,. Lu et al, (2004) JBC 279: 2856 to 2865; Lu et al, (2005) JBC 280: 19665 to 19672 ,. Marvin et al, (2005) Acta Pharmacologica Sinica 26: 649 to 658;. Marvin et al., (2006 ) Curr Opin Drugs Disco Develop 9: 184-193; Shen et al, (2007) Methods J Immun 218: 65 to 74;. Wu et al, (2007) Nat Biotechnol 11: 1290 to 1297, DiMasi et al, (2009 ) J Mol Biol 393: 672 to 692, and Michaelson et al, (2009) mAbs 1: 128 to 141.
[0338] The bispecific molecules of the present invention can be prepared by conjugating the constituent specificities of binding, using the methods known in the art. For example, each bispecific molecule binding specificity can be generated separately and then conjugated to one another. When binding specificities are proteins or peptides, a variety of coupling or crosslinking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5 '-ditiobis (2-nitrobenzoic) (DTNB), o-phenylenedimleimide (oPDM), N propionate - succinimidyl-3- (2-pyridyldithium) (SPDP), and sulfosuccinimidyl 4 - (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al, (1984) J. Exp Med. 160: 1686, Liu et al, (1985) Proc Natl Acad Sci US 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78: 118 to 132, Brennan et al, (1985) Science 229: 81 to 83), and Glennie et al, (1987) J. Immunol. 139: 2367 to 2375). Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).
[0339] [00339] When the binding specificities are antibodies, they can be conjugated to the sulfhydryl bond of the Hinge Terminal C regions of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, for example, one, prior to conjugation.
[0340] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb mAb x, mAb x Fab, Fab x F (ab ') 2 or ligand x Fab of the fusion protein. A bispecific molecule of the present invention can be a single chain molecule comprising a single chain antibody and a binding determinant, or a bispecific single chain molecule comprising two binding determinants. Bispecific molecules can comprise at least two single-stranded molecules. Methods for preparing bispecific molecules are described, for example, in US Patent No. 5260203, US Patent Number 5,455,030, US Patent Number 4,881,175, US Patent Number 5,132,405, US Patent Number 5,091,513, US Patent Number 5,476 .786, US Patent number 5,013,653, US Patent number 5,258,498; and U.S. Patent Number 5,482,858.
[0341] [00341] The attachment of bispecific molecules to their specific objectives can be confirmed through, for example, enzyme linked immuno-sorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (eg growth inhibition) , or Western Blot assay. Each of these assays detects, generally in the presence of protein-antibody of particular interest employing a labeled reagent (e.g., an antibody) specific to the complex of interest.
[0342] [00342] In another aspect, the present invention provides multivalent compounds that comprise at least two identical or different fragments of the antibodies of the present invention for HER3 binding. The antibody fragments can be linked together by means of fusion or covalent or non-covalent binding protein. The tetravalent compounds can be obtained, for example, by cross-linking the antibodies. The antibodies of the present invention with an antibody that binds to the constant regions of the antibodies of the present invention, for example, the hinge or Fc region. The Trimerization domain are described for example in EP 1012280B1 Borean. Pentamerization modules are described for example in PCT / EP97 / 05897.
[0343] [00343] In one embodiment, a biparatopic / bispecific binds to amino acid residues within domain 2 and domain 4 of HER3.
[0344] [00344] In another embodiment, the present invention relates to dual-function antibodies in which a single monoclonal antibody has been modified in such a way that the antigen-binding site binds to more than one antigen, such as an antibody that the dual function binds to both HER3 and another antigen (for example, HER1, HER2 and HER4). In another embodiment, the present invention relates to an antibody that targets dual function antigens having the same conformation, for example, an antigen that has the same conformation as HER3 in the "closed" or "inactive" state. Examples of antigens, with the same conformation as HER3 in the "closed" or "inactive" state include, but are not limited to, HER1 and HER4. In this way, an antibody can bind to the dual function of both HER3 and HER1, HER3 and HER4, or HER1 and HER4. The double binding specificity of the dual function antibody may further translate into dual activity, or inhibition of activity. (See, for example, Jenny Bostrom et al, (2009) Science: 323; 1610 to 1614.). Antibodies with Extended Half-Life
[0345] [00345] The present invention provides antibodies that specifically bind to the HER3 protein that have a prolonged half-life in vivo.
[0346] [00346] Many factors can affect the half-life of a protein in vivo. For example, filtration of the kidneys, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenic responses (for example, neutralizing protein by means of antibodies and absorption by macrophages and dendritic cells). A variety of strategies can be used to extend the half-life of the antibodies of the present invention. For example, by chemical bonding with polyethylene glycol (PEG), recode PPOR EXAMPLE, antibody scaffolding, polysialic acid (PSA), hydroxyethyl starch (HES), linker binding albumin, and carbohydrate shields; through the genetic fusion of proteins that bind to whey proteins, such as albumin, IgG, FcRn, and transfer, by coupling (genetically or chemically) to other binding portions that bind to whey proteins, such as nanobodies, DARPins, avimeros, afficorpos and anticalins Fabs; through genetic fusion to rPPOR EXAMPLE, albumin, albumin domain, albumin-binding proteins, and Fc; or through incorporation into nanoparticles, slow release formulations or medical devices.
[0347] [00347] In order to prolong the circulation in the serum of antibodies in vivo, inert polymer molecules, such as high molecular weight PEG can be linked to the antibodies or a fragment thereof, with or without a multifunctional linker, either through the specific site of conjugation of PEG with an N or C terminal of the antibodies or via the epsilan-amino groups present in lysine residues. To pegylate an antibody, the antibody, or a fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester or an aldehyde derivative of PPOR EXAMPLE, under conditions where one or more PEG groups have become attached to the antibody or antibody fragment. Pegylation can be carried out by means of an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a reactive water-soluble polymer analog). As used in the present invention, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to transform other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene -glycol-maleimide. In certain embodiments, the antibody to be treated with PEG is an aglycosylated antibody. A linear or branched derivatization of the polymer that results in a minimal loss of biological activity will be used. The degree of conjugation can be carefully monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules with antibodies. Unreacted PEG can be separated from the PEG-conjugated antibody by size exclusion or by ion exchange chromatography. PEG-derived antibodies can be tested for binding activity, as well as for in vivo efficacy, using methods well known to those skilled in the art, for example, by means of the immunoassays described in the present invention. Methods for pegylation proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
[0348] [00348] Other modified pegylation technologies include the reconstitution of directed orthogonal chemical engineering technology (recode PEG), which incorporates chemically specified side chains into biosynthetic proteins through a system that includes tRNA synthetase and reconstituted tRNA. This technology allows the incorporation of more than 30 new amino acids in biosynthetic proteins in E. coli, yeast and mammalian cells. The tRNA incorporates a non-native amino acid anywhere an amber codon is positioned, the conversion of amber from a stop codon to one that signals the incorporation of the chemically specified amino acid.
[0349] [00349] Recombinant pegylation technology (rPEG) can also be used for the extension of serum half-life. This technology genetically involves fusing 300 to 600 amino acids from the unstructured tail protein to an existing pharmaceutical protein. Since the apparent molecular weight of the protein of such an unstructured chain is about 15 times greater than its actual molecular weight, the serum half-life of the protein is much greater. In contrast to traditional pegylation, which requires chemical conjugation and repurification, the manufacturing process is very simplified and the product is homogeneous.
[0350] [00350] Polyisialization is another technology, which uses the natural polymer, polyasylic acid (PSA) to prolong the active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and peptide therapeutic drug delivery, polysialic acid provides a protective microenvironment in conjunction. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. The PSA polymer is naturally found in the human body. It was adopted by certain bacteria that evolved over millions of years to coat its walls with it. These naturally polysylylated bacteria were able, by virtue of molecular mimicry, to outwit the body's defense system. PSA, the ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when associated with proteins, since it is chemically identical to PSA in the human body.
[0351] [00351] Another technology includes the use of starch from hydroxyethyl derivatives ("HES") associated with antibodies. HES is a modified natural polymer derived from waxy corn starch and can be metabolized by the body's enzymes. HES solutions are generally administered to replace the deficient blood volume and to improve the rheological properties of the blood. HESylation of an antibody allows the prolongation of the circulation half-life, increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increase in biological activity. By varying the different parameters, such as the molecular weight of HES, a wide range of conjugated HES antibodies can be customized.
[0352] [00352] Antibodies having a longer half-life in vivo can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) for an IgG constant domain, or a binding fragment therefrom (preferably , an Fc fragment or Fc hinge domain). See, for example, International Publication No. WO 98/23289; International Publication No. WO 97/34631, and U.S. Patent No. 6,277,375.
[0353] [00353] In addition, antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo, or to have a longer half-life in vivo. The techniques are well known in the art, see, for example, International Publication No. WO 93/15199, WO 93/15200, and WO 01/77137 and European Patent No. EP 413622.
[0354] [00354] The HER3 antibody or a fragment thereof can also be fused with one or more human serum albumin (HSA), polypeptides, or a part of it. HSA, a 585 amino acid protein in its mature form, is responsible for a significant proportion of serum osmotic pressure and also functions as a carrier for endogenous and exogenous ligands. The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a carrier and transporter of polypeptides in vivo. The use of albumin as a component of an albumin fusion protein as a carrier for various proteins has been suggested in WO 93/15199, WO 93/15200 and EP 413 622. The use of N-terminal fragments of HSA for fusions with polypeptides have also been proposed (EP 399 666). Therefore, by genetic fusion or chemically or conjugating the antibodies or their fragments to the albumin, it can stabilize or increase the useful life period, and / or to retain the activity of the molecule, for prolonged periods in solution, in vitro and / or in vivo.
[0355] [00355] Fusion of albumin to another protein can be achieved by genetic manipulation, such that the DNA encoding HSA, or a fragment thereof, is joined to the DNA encoding the protein. A suitable host is then transformed or transfected with the fused nucleotide sequences, arranged so as to form a suitable plasmid to express a fusion polypeptide. Expression can be carried out in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo, for example from a transgenic organism. Additional methods relating to HSA fusions can be found, for example, in WO 2001077137 and WO 200306007, in the present invention incorporated by reference. In a specific embodiment, the expression of the fusion protein is carried out in mammalian cell lines, for example, the CHO cell lines. Binding altered differentials of an antibody to a receptor at low or high pHs is also contemplated to be within the scope of the present invention. For example, the affinity of an antibody can be modified in such a way that it remains bound to its receptor, at a low pH, for example, the low pH within a lyzozome, modifying the antibody to include additional amino acids, such as a histine in a CDR of the antibody (See, for example, Tomoyuki Igawa et al (2010) Nature Biotechnology ,. 28, 1203-1207). Antibody conjugates
[0356] The present invention provides antibodies or fragments thereof that specifically bind to a recombinantly fused or chemically conjugated HER3 protein (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof) preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate proteins of fusion. In particular, the present invention provides fusion proteins comprising an antibody fragment in the present invention described (for example, a Fab, Fd fragment, Fv fragment, F (ab ') fragment 2 fragments, a VH domain, a VH CDR, a VL or a VL CDR domain) and a heterologous protein, polypeptide or peptide. Methods for fusing or conjugating proteins, polypeptides or peptides, to an antibody or an antibody fragment are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent No. EP 307434 and EP 367166; International Publication No. WO 96/04388 and WO 91/06570; Ashkenazi et al ,. (1991) Proc. Natl. Acad. U.S. Sci. 88: 10535-10539 ,. Zheng et al, (1995) J. Immunol. 154: 5590-5600; and Vil et al, (1992) Proc .. Natl. Acad. U.S. Sci. 89: 11337 - 11341.
[0357] [00357] Additional fusion proteins can be generated using gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA scrambling can be used to alter the activities of the antibodies of the present invention or fragments thereof (for example, antibodies or fragments thereof with higher affinities and lower dissociation rates). see, in general, U.S. Patent No. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, (1997) Curr. Opinion Biotechnol. 8: 724-33; Harayama, (1998) Trends Biotechnol. 16 (2): 76 to 82; Hansson et al, (1999) J. Mol. Biol. 287: 265-76, and Lorenzo and Blasco, (1998) Biotechniques 24 (2): 308 to 313 (each of these patents and publications are hereby incorporated by reference in their entirety). The antibodies or their fragments, or the encoded antibodies or their fragments, can be altered by being subjected to random muetiquetaênese by error-prone PCR, random insertion of nucleotides or other methods prior to recombination. A polynucleotide that encodes an antibody or a fragment thereof that specifically binds to an HER3 protein can be recombined with one or more components, motifs, profiles, pieces, domains, fragments, etc., of one or more heterologous molecules.
[0358] [00358] In addition, the antibodies or fragments thereof can be fused to the marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the amino acid sequence marker is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., (1989) Proc. Natl. Acad. U.S. Sci. 86: 821 to 824, for example, hexahistidine provides for convenient purification of the fusion protein. Other peptide brands useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., (1984) Cell 37: 767 ), and the "flag" tag.
[0359] [00359] In another embodiment, the antibodies of the present invention or fragments thereof are conjugated to a diagnostic or detectable agent. Such antibodies may be useful for monitoring or prognosis of the appearance, development, progression and / or the severity of a disease or disorder, as part of a clinical testing procedure, such as determining the effectiveness of a particular therapy. Such diagnosis and detection can be achieved by coupling the antibody to detectable substances include, but are not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidinlbiotin and avidin / biotin; fluorescent materials, such as, but not limited to, umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol, bioluminescent materials, such as but not limited to, luciferase, luciferin, and aquorin; radioactive materials, such as, but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho , 90Y, 47Sc, 186Re, 188Re, 142 Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin and positron emitting metals using various emission tomographs of positrons, and non-radioactive paramagnetic metal ions.
[0360] [00360] The present invention further encompasses the uses of antibodies or fragments thereof conjugated to a therapeutic moiety. An antibody or fragment can be conjugated to a therapeutic moiety such as a cytotoxin, for example, a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, for example, alpha emitters. A cytotoxin, or cytotoxic agent, includes any agent that is harmful to cells.
[0361] [00361] In addition, an antibody or fragment can be conjugated to a therapeutic moiety or drug molecule that modifies a given biological response. Radicals or therapeutic portions of drugs are not to be interpreted as limited to classical therapeutic chemical agents. For example, the drug molecule can be a protein, peptide, or polypeptide having a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth, platelet-derived growth, tissue plasminogen activating factor, an apoptosis agent, an anti-angiogenic agent, or a biological response modifier, such as, for example, a lymphokine. In one embodiment, the anti-HER3 antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (for example, an immunosuppressant), or a radiotoxin. Such conjugates are referred to in the present invention as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins". A cytotoxin, or cytotoxic agent, includes any agent that is harmful to (for example, kills) cells. Examples include taxon, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, atinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or counterparts thereof. Additional therapeutic agents include, for example, anti-metabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablation agents (for example, mecloretamine, thioepa chloraxnbucil, meiphalan, carmustine (BSN ) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis dichlorodiamine-platinum (II) with cisplatin (DDP), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin) , datinomycin (formerly atinomycin), bleomycin, mitramycin and anthramycin (AMC)), and anti-mitotics (for example, agents, vincristine and vinblastine). (See, for example, Seattle Genetics US20090304721).
[0362] [00362] Other examples of therapeutic cytotoxins that can be conjugated to an antibody of the present invention, include, calicheamicin duocarmicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (MylotargTm; Wyeth-Ayerst).
[0363] [00363] Cytoxins can be conjugated to the antibodies of the present invention using the binding technology available in the art. Examples of types of linkers that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and containing the linker peptides. A linker can be chosen, that is, for example, susceptible to low pH cleavage within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases, preferably expressed in tumor tissue, such as cathepsins (for example, cathepsins). cathepsins B, C, D).
[0364] [00364] For an additional discussion of types of cytotoxins, ligands and methods for conjugating therapeutic agents to antibodies, see also Saito et al., (2003) Adv. Drug Deliv. Rev. 55: 199 to 215; Trail et al, (2003) Cancer Immunol .. Immunother. 52: 328 to 337; Payne (2003) Cancer Cell 3: 207 to 212; Allen, (2002) Nat. Rev. Cancer 2: 750 to 763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3: 1089 to 1091; Senter and Springer, (2001) ADV. Drug Deliv. Rev. 53: 247 to 264.
[0365] [00365] The antibodies of the present invention can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use in diagnosis or therapy include, but are not limited to, iodinel31, indium111, yttrium90 and lutetium177. The method for preparing radioimmunoconjugates is established in the art. Examples of radioimmunoconjugates are commercially available, including ZevalinTM (DEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the present invention. In certain embodiments, the chelating agent is macrocyclic 1,4,7,10-tetraazacyclododecane-N, N ', N' ', N' "- tetraacetic acid (DOTA), which can be linked to the antibody via a molecule Such ligand molecules are generally known in the art and described in Denardo et al., (1998), Clin Cancer Res. 4 (10): 2483 to 90; Peterson et al, (1999) Bioconjug. Chem. 10 (4 ): 553 to 7, and Zimmerman et al, (1999) Nucl .. Med. Biol. 26 (8): 943 to 50, each incorporated by reference in its entirety.
[0366] [00366] The techniques for conjugating therapeutic portions of antibodies are well known, see, for example, Arnon et al. "Monoclonal Antibodies for Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (Eds.), Pp 243 to 56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies for Drug Delivery", in Controlled Drug Delivery (2nd ed.), Robinson et al .. (Eds.), Pp 623 to 53 (Marcel Dekker, Inc., 1987); Thorpe, "Antibodies Carriers of Citotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al. (Eds.), Pp 475 to 506 (1985), "Anlysis, Results and Future prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (Eds.), Pp 303-16 (Academic Press 1985), and Thorpe et al., (1982) Immunol. Rev. 62: 119 to 58.
[0367] [00367] Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polyvinyl chloride, polystyrene or polypropylene. Combinations of antibodies
[0368] [00368] In another aspect, the present invention relates to HER3 Antibodies, or fragments thereof of the present invention used with other therapeutic agents, such as antibodies, inhibitors of other small molecules, mTOR inhibitors and PI3Kinase inhibitors. Examples include, but are not limited to, the following:
[0369] [00369] HER1 Inhibitors: HER3 Antibodies or fragments thereof can be used with HER1 inhibitors, which include, but are not limited to, Matuzumab (EMD72000), Erbitux ® Cetuximab / (Imclone), Vectibix ® / Panitumumab (Amgen ), mAb 806, and Nimotuzumab (TheraCIM), Iressa ® / Gefitinib (AstraZeneca), IC-1033 (PD183805) (Pfizer), lapatinib (GW-572016) (GlaxoSmithKline), Tykerb® / lapatinib ditosilato (SmithKlinee ® / erlotinib HCL (OSI-774) (OSI Pharma) and PKI-166 (Novartis), and sold under the trademark Tovok ® by Boehringer Ingelheim).
[0370] [00370] HER2 inhibitors: HER3 antibodies or fragments thereof can be used with HER2 inhibitors, which include, but are not limited to, pertuzumab (sold under the trademark Omnitarg ®, by Genentech), trastuzumab (sold under the trademark of Herceptin ® by Genentech / Roche), MM-111, neratinib (also known as HKI-272, and described PCT Publication No. WO 05/028443), lapatinib or lapatinib ditosylate (sold under the Tykerb ® trademark by GlaxoSmithKline.
[0371] [00371] HER3 Inhibitors: HER3 Antibodies or fragments thereof can be used with HER3 inhibitors, which include, but are not limited to, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888 ( Amgen), AV-203 (Aveo), MEHD7945A (Genentech), and the small molecules that inhibit HER3.
[0372] [00372] HER4 Inhibitors: HER3 Antibodies or fragments thereof can be used with Her4 inhibitors.
[0373] [00373] PI3K Inhibitors: An HER3 Antibodies or fragments thereof can be used with PI3 kinase inhibitors, which include, but are not limited to, (Also known as GDC 0941 and described in PCT Publication No. WO 09/036082 and WO 09/055730), (Also known as BEZ 235 or NVP-BEZ 235, and described in PCT Publication No. WO 06/122806), and BMK120 BYL719.
[0374] [00374] mTOR inhibitors: HER3 Antibodies or fragments thereof can be used with mTOR inhibitors, which include, but are not limited to, Temsirolimus (sold under the trade name Torisel ® by Pfizer), ridaforolimus (formally known as deferolimus, (1R, 2R, 4S) -4 - [(2R) -2 [(1R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R) -1,18-dihydroxy-19, 30-dimethoxy-15, 17,21,23, 29,35-hexamethyl-2-pentaoxo, 3,10,14,20-11, 36-dioxa-4-azathricycle [30.3.1.04.9] hexatriaconta-16, 24,26,28-tetraen-12-yl] propyl] -2-methoxycyclo dimethylphosfinate, also known as Deforolimus, AP23573 and MK8669 (Ariad Pharm.), And described in PCT Publication No. WO 03/064383), everolimus (RAD001) (sold under the trade name of ® Afinitor Novartis), one or more therapeutic agents can be administered either simultaneously, or before or after the administration of an antibody or a fragment thereof HER3 of the present invention. Methods of producing antibodies of the present invention (I) nucleic acids encoding antibodies
[0375] The present invention provides substantially purified nucleic acid asmolecules encoding the polypeptides comprising the HER3 Antibody binding chain segments or domains described above. Some of the nucleic acids of the present invention comprise the nucleotide sequence encoding the HER3 antibody variable region of the heavy chain, and / or the nucleotide sequence encoding the variable region of the light chain. In a specific embodiment, the nucleic acid molecules are those identified in Table 1. Some other nucleic acid molecules of the present invention comprise nucleotide sequences that are substantially identical (for example, at least 65, 80%, 95% or 99 %) with the nucleotide sequences of those identified in Table 1. When expressed from appropriate expression vectors, polypeptides encoded by these polynucleotides are capable of exhibiting HER3 antigen-binding capacity.
[0376] [00376] Also provided in the present invention are polynucleotides that encode at least one CDR region and, generally, all three heavy or light chain CDR regions of the HER3 binding antibody set out above. Some other polynucleotides encode all or substantially the entire sequence of the variable region of the heavy chain and / or the light chain of the HER3 binding antibody set out above. Due to the degeneracy of the code, several nucleic acid sequences that encode each in the immunoglobulin amino acid sequences.
[0377] [00377] The nucleic acid molecules of the present invention can encode both the variable region and an antibody constant region. Some of the nucleic acid sequences of the present invention comprise the nucleotides encoding the mature heavy chain sequence of the variable region, which is substantially identical (for example, at least 80%, 90% or 99%) to the mature heavy chain sequence the variable region of a set of HER3 antibodies shown in Table 1. Some other nucleic acid sequences comprising nucleotides encoding a mature light chain sequence of the variable region, which is substantially identical (for example, at least 80%, 90 % or 99%) for the mature light chain sequence of the variable region of an HER3 antibody shown in Table 1.
[0378] [00378] Polynucleotide sequences can be produced by re-synthesizing solid-phase DNA or by PCR mutagenesis of an existing sequence (for example, the sequences as described in the Examples below) encoding a HER3, or the fragment thereof. Direct chemical synthesis of nucleic acids can be performed using methods known in the art, such as the phosphotriester method of Narang et al., (1979) Meth. Enzymol. 68: 90, the phosphodiester method of Brown et al, (1979) Meth. Enzymol. 68: 109, the Beaucage et al, (1981) Tetra method of diethylphosphoramiditis. Lett, 22: 1859; and the solid support method of U.S. Patent No. 4,458,066. mutations that introduce a polynucleotide sequence by PCR can be performed as described in, for example, PCR Technology: Principies and Applications for DNA Amplification, HA Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; . Mattila et al, (1991) Nucleic Acids Res. 19: 967; and Eckert et al, (1991) PCR Methods and Applications 1: 17.
[0379] [00379] Also provided in the present invention are the expression vectors and host cells for producing HER3-binding antibodies described above. Different expression vectors can be used to express the polynucleotides that encode HER3 Antibody binding chains or binding fragments. Both virus-based and non-viral expression vectors can be used to produce antibodies to a mammalian host cell. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for the expression of a protein or RNA, and human artificial chromosomes (see, for example, Harrington et al., (1997) Nat Genet 15: 345) . For example, non-viral vectors useful for the expression of HER3 polynucleotides and binding polypeptides in mammalian (e.g. human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C, ( Invitrogen, San Diego, CA), the MPSV vectors, and numerous other vectors known in the art for the expression of other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papillama viruses, Epstein Barr HBP viruses, vectors of vaccinia virus and Semliki Forest virus (SFV). See, Brent et al, (1995) supra; . Smith, Annu. Rev. Microbiol. 49: 807, and Rosenfeld et al, (1992) Cell 68: 143.
[0380] [00380] The choice of the expression vector depends on the intended host cells, in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides that encode an antibody binding chain or the HER3 fragment. In some embodiments, an inducible promoter is used to prevent expression of inserted sequences except under conditions of induction. Inducible promoters include, for example, arabinose, lacZ, the metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under non-inductive conditions without polarization of the population of coding sequences whose expression products are well tolerated by host cells. In addition to promoters, other regulatory elements may also be necessary or desired for the efficient expression of an antibody binding chain or the HER3 fragment. These elements typically include an ATG initiation codon and binding site to the adjacent ribosome or other sequences. In addition, the efficiency of expression can be increased by the inclusion of promoters appropriate for the cellular system in use (see, for example, Scharf et al, (1994) Results Probl cellular Differ 20: 125, E Bittner et al., (1987 ) Meth. Enzymol., 153: 516). For example, the SV40 enhancer or the CMV enhancer can be used for the purpose of increasing expression in mammalian host cells.
[0381] The expression vectors can also provide a position secretion signal sequence to form a fusion protein with polypeptides encoded by the sequences inserted into the HER3 binding antibodies. Most often, the sequences inserted into the HER3 binding antibodies are linked to a sequence of signals prior to inclusion in the vector. The vectors to be used to receive sequences encoding light chain HER3 binding antibodies and the heavy chain variable domains sometimes also encode constant regions, or parts of them. These vectors allow the expression of variable regions, such as fusion proteins with constant regions as soon as they lead to the production of intact antibodies or fragments thereof. Typically, these constant regions are human.
[0382] [00382] Host cells to harbor and express HER3 antibody binding chains can be prokaryotic or eukaryotic. E. coli is a prokaryotic host useful for the cloning and expression of the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various species of Pseudomonas. In these prokaryotic hosts, one can also make expression vectors, which normally contain expression control sequences compatible with the host cell (for example, an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a lambda phage promoter system. Promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, to initiate and complete transcription and translation. Other microbes, such as yeasts, can also be employed to express HER3-binding polypeptides of the present invention. Insect cells in conjunction with baculovirus vectors can also be used.
[0383] In some preferred embodiments, mammalian host cells are used to express and produce the HER3-binding polypeptides of the present invention. For example, they may be a hybridoma cell line that expresses endogenous immunoglobulin genes (for example, the hybridoma myeloma clone 1D6.C9 as described in the examples) or a mammalian cell line harboring an exogenous expression vector (for example, example, to myeloma SP2 / 0 cells exemplified below). These include any mortal or normal or abnormal, normal, immortal animal or human cell. For example, a number of suitable host cell lines capable of intact secretory immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian cell and tissue culture to express polypeptides is discussed in general, for example, Winnacker, DE GENES DE CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include the sequences expression control, such as an origin of replication, a promoter and an enhancer (see, for example, Queen et al., (1986) Immunol. Rev. 89: 49 to 68), and the processing locations required for information, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. These expression vectors normally contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, the specific cell type, the specific stage, and / or modulable or adjustable. Useful promoters include, but are not limited to, the metallothionein promoter, the main constituent late adenovirus promoter, the MMTV dexamethasone-inducible promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the CMV inducible tetracycline promoter (such as the immediate human early CMV promoter), the constitutive CMV promoter, and the promoter-enhancer combinations known in the art.
[0384] [00384] The methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts. (See generally, Sambrook, et al., Supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation: nucleic acid conjugates, naked DNA, artificial virions, fusion with the structural protein VP22 of the herpes virus (Elliot and O'Hare, (1997) Cell 88: 223), a DNA absorption enhancing agent, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression is often to be desired. For example, cell lines that stably express HER3 antibody binding chains or binding fragments can be prepared using the expression vectors of the present invention that contain viral replication origins or endogenous expression elements and a selectable marker gene. After the introduction of the vector, the cells can be left to grow for 1 to 2 days in an enriched medium before being switched to the selective media. The purpose of the selection marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the sequences introduced in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type. (Ii) Production of monoclonal antibodies of the present invention
[0385] [00385] Monoclonal antibodies (mAb) can be produced using a variety of techniques, including, for example, the conventional monoclonal antibody methodology, the Kohler and Milstein hybridization somatic cell pattern, (1975) Nature 256: 495. Many techniques for the production of monoclonal antibodies can be employed, for example, viral or oncogenic transformation of B lymphocytes.
[0386] [00386] An animal system for preparing hybridomas is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for the isolation of immunized splenites for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known.
[0387] [00387] Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain the non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions, using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, murine CDR regions can be inserted into a human structure, using methods known in the art. See, for example, U.S. Patent No. 5,225,539 to Winter, and U.S. Patent No. 5,530,101; 5585089; 5,693,762 and 6,180,370 to Queen et al.
[0388] [00388] In a given embodiment, the antibodies of the present invention are human monoclonal antibodies. Such human monoclonal antibodies directed against HER3 can be generated using transgenic or trans-chromosomal mice that transport parts of the human immune system instead of the mouse system. These transgenic and trans-chromosomal mice include mice in the present invention referred to as mice and AcMHu km mice, respectively, and are referred to in the present invention collectively as "human Ig mice."
[0389] [00389] The AcMHu ® mouse (Medarex, Inc.) contains immunoglobulin from the human minilaci gene that encodes the unorganized human heavy chain (μ and γ) and the k light chain immunoglobulin sequences, along with specific mutations that inactivate the μ endogenous and κ sites of the chain (see, for example, Lonberg et al, (1994) Nature 368 (6474): 856 to 859). Consequently, mice show reduced expression of mouse or k IgM, and in response to immunization, introduced human and light heavy chain transgenes undergo switching and somatic mutation to generate high human affinity monoclonal IgGK (Lonberg et al. , (1994) supra; revised in Lonberg, (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg and Huszar, (1995) Intern Rev. Immunol.13: 65 to 93, and Harding and Lonberg, (1995) Ann NY Acad. Sci. 764: 536 to 546). The preparation and use of AcMHu mice, and the genomic modifications carried by these mice, is further described in Taylor et al, (1992) Nucleic Acids Research 20: 6287 to 6295; Chen et al, (1993) International Immunology 5: 647 to 656 ,. Tuaillon et al, (1993) Proc. Natl. Acad. U.S. Sci. 94: 3720 to 3724; Choi et al, (1993) Nature Genetics 4: 117 to 123; . Chen et al, (1993) EMBO J. 12: 821 to 830; Tuaillon et al, (1994) J. Immunol. 152: 2912 to 2920; Taylor et al, (1994) International Immunology 579 to 591, E Fishwild et al, (1996) Nature Biotechnology 14: 845 to 851, the contents of all of which are in the present invention expressly incorporated by reference in their entirety. See, further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299 and 5,770,429, all to Lonberg and Kay, U.S. Patent No. 5,545,807 to Surani et al. PCT Publication No. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO 99/45962, all by Lonberg and Kay, and PCT Publication No. WO 01/14424 by Korman et al.
[0390] [00390] In another embodiment, the human antibodies of the present invention can be raised using a mouse that carries human immunoglobulin sequences in transgenes and transcromosomes such as a mouse that carries a human heavy chain transgene and a human light chain. Such mice, in the present invention referred to as "KM mice", are described in detail in PCT Publication WO 02/43478 to Ishida et al.
[0391] [00391] Furthermore, alternative systems of transgenic animals that express human immunoglobulin genes are available in the art and can be used to increase the binding of HER3 Antibodies of the present invention. For example, an alternative transgenic system referred to as Xenocamundongo (Abgenix, Inc.) can be used. Such mice are described in, for example, U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598, 6, 150584 and 6162963 to Kucherlapati et al.
[0392] [00392] In addition, alternative systems of transchromosomal animals that express human immunoglobulin genes are available in the art and can be used to increase the binding of HER3 Antibodies of the present invention. For example, mice that carry both a transchromosome human heavy chain and a human trancromosome light chain, referred to as "TC mice" can be used, such mice are described in Tomizuka et al, Proc (2000). Natl. Acad. Sci. U.S. 97: 722-727. In addition, human transchromosomes carrying light and heavy chains have been described in the art (Kuroiwa et al., (2002) Nature Biotechnology 20: 889-894) and can be used to increase the binding of HER3 Antibodies of the present invention.
[0393] [00393] The human monoclonal antibodies of the present invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such display phage methods for isolating human antibodies are established in the art and described in the examples below. See, for example: U.S. Patent Nos. 5,223,409; 5,403,484, and 5,571,698 by LDNAer et al; U.S. Patents Nos. 5,427,908 and 5,580,717 to Dower et al; U.S. Patents Nos. 5,969,108 and 6,172,197 to McCafferty et al, and U.S. Patent No. .... 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.
[0394] [00394] The human monoclonal antibodies of the present invention can also be prepared using SCID mice in which the immune cells have been reconstituted in such a way that a human antibody response can be generated by immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al. (Iii) Fc structure or engineering
[0395] [00395] The antibody engineering of the present invention includes those in which modifications have been made to structural residues within VH and / or VL, for example, to improve the properties of the antibody. Typically such changes in structure are made to decrease the immunogenicity of the antibody. For example, an approach is "removed" from one or more structure residues with the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain residues of the structure that differ from the germline sequence from which the antibody is derived. These residues can be identified by comparing the structural antibody sequences to the germline sequences from which the antibody is derived. In order to return the sequences of the region of the structure to its germline configuration, somatic mutations can be "removed" with the germline sequence, for example, muetiquetaênese directed to the site. Such "repudiated" antibodies are also intended to be encompassed by the present invention.
[0396] [00396] Another type of modification involves the mutant structure of one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "de-immunization" and is described in more detail in U.S. Patent Publication No. 20030153043 by Carr et al.
[0397] [00397] In addition or alternatively, modifications made to the framework or CDR regions, the antibodies of the present invention can be modified to include modifications within the Fe region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, binding Fc receptor, and / or cell antigen-dependent cytotoxicity. In addition, an antibody of the present invention can be chemically modified (for example, one or more chemical groups can be attached to the antibody), or be modified in order to alter its glycosylation, again to alter one or more functional properties of the antibody . Each of these modalities is described in detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.
[0398] [00398] In one embodiment, the CH1 hinge region is modified in such a way that the number of cysteine residues in the hinge region is altered, for example, increased or decreased. This approach is described later in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the CH1 hinge region is changed to, for example, facilitate the monetization of light and heavy chains or to increase or decrease the stability of the antibody.
[0399] [00399] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fc hinge fragment in such a way that the antibody has hindered the binding of the relative Staphylococcyl A protein (SpA) to the native SpA domain of the Fc hinge. binding. This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al.
[0400] [00400] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced by a different amino acid residue such that the antibody has an altered affinity for an effector linker, but retains the antigen-binding capacity of the parent antibody. The effector ligand to which the affinity changes can be, for example, an Fc receptor or complement component C1. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.
[0401] [00401] In another embodiment, one or more amino acids selected from the amino acid residues can be replaced with a different amino acid residue such that the C1q antibody altered binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al.
[0402] [00402] In another embodiment, one or more amino acid residues are altered to alter the ability of the antibody to fix the complement. This approach is described later in PCT Publication WO 94/29351 by Bodmer et al.
[0403] [00403] In yet another embodiment, the Fc region is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the antibody's affinity for an Fcy receptor by modifying a or more amino acids. This approach is described later in PCT Publication WO 00/42072 by Presta. In addition, the human IgG1 binding points for FcγRI, FcyRII, and FcyRIII FcRn have been mapped and improved linked variants have been described (see Shields et al., (2001) J. Biol. Chen. 276: 6591 to 6604) .
[0404] [00404] In yet another embodiment, the glycosylation of an antibody is modified. For example, an antibody can be made non-glycolysed (that is, the antibody lacks glycosylation). Glycosylation can be changed, for example, in order to increase the antibody's affinity for "antigen". Such carbohydrate modifications can be achieved by, for example, altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more glycosylation sites from the variable region of the structure, thereby eliminating glycosylation at that location. Such non-glycolization may increase the antibody's affinity for the antigen. Such an approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
[0405] [00405] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased GlcNAc bisector structures. Such altered glycosylation patterns have been shown to increase the capacity of ADCC antibodies. Such carbohydrate modifications can be achieved by, for example, expressing the antibody in a host cell with the present invention of altered glycosylation. Cells with altered glycosylation machines have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, to thereby produce an antibody with altered glycosylation. For example, EP 1176195 by Hang et al. describes a cell line with a functionally corrupted FUT8 gene, which encodes a fucosyl transferase, in such a way that the antibodies expressed in such a hypofucosylation exposure of the cell line. PCT Publication WO 03/035835 describes by Presta a variant of the CHO cell line, Lecl3 cells, with reduced ability to attach fucose to carbohydrates linked to Asn (297) -, also results in hypofucosylation of antibodies expressed in the host cell (see also Shields et al., (2002) J. Biol. Chem. 277: 26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines modified to express glycoprotein by modifying glycosyl transferases (e.g., beta (1,4) -N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in engineered cell lines have greater bisecting structures GlcNAc o which results in increased ADCC activity of the antibodies (see also Umana et al., (1999) Nat. Biotech. 17: 176 to 180).
[0406] [00406] In another embodiment, the antibody is modified to increase its biological half-life. Several approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be changed within the CH1 or the CL region to contain a rescue receptor binding epitope taken from two loops of a CH2 domain of the IgG Fc region, such as as described in US Patent Nos. 5,869,046 and 6,121,022 to Presta et al. (Iv) Engineering methods of altered antibodies
[0407] [00407] As discussed above, HER3 binding antibodies having the VH and VL sequences or full length heavy and light chain sequences shown in the present invention can be used for the purpose of creating new HER3 binding antibodies by modification of the full-length heavy chain and / or light chain sequences, VH and / or VL sequences, or the constant region (s) attached to it. Thus, in another aspect of the present invention, the structural characteristics of an HER3-binding antibody of the present invention are used to create structurally related HER3-binding antibodies that retain at least one functional property of the antibodies of the present invention, such as binding to human HER3 and also inhibiting one or more functional properties of HER3 For example, one or more CDR regions of antibodies of the present invention, or their mutations, can be combined recombinantly with known structural regions and / or other CDRs to create additionally, recombinantly engineered, the HER3-binding antibodies of the present invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the VH and / or VL sequences provided in the present invention, or one or more CDR regions thereof. To create the engineered antibody, which is not necessary to effectively prepare (ie expressed as a protein), an antibody that has one or more of the VH and / or VL sequences provided in the present invention, or one or more CDR regions of the themselves. Instead, the information contained in the sequence (s) is used as starting material to create a "second generation" sequence (s) derived from the original sequence (s) and then , the "second generation" of sequence (s) is prepared and expressed as a protein.
[0408] [00408] Therefore, in another embodiment, the present invention provides a method for the preparation of an HER3-binding antibody that consists of: a variable sequence of heavy antibody chain having a region of CDR1 sequence selected from the group consisting of SEQ ID NOs: 2, 8, 20, 26, 38, 44, 56, 62, 74, 80, 92, 98, 110, 116, 128, 134, 146, 152, 164, 170, 182, 188 , 200, 206, 218, 224, 236, 242, 254, 260, 272, 278, 290, 296, 308, 314, 326, 332, 344, 350, 362, and 368; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 3, 9, 21, 27, 39, 45, 57, 63, 75, 81, 93, 99, 111, 117, 129, 135, 147, 153 , 165, 171, 183, 189, 201, 207, 219, 225, 237, 243, 255, 261, 273, 279, 291, 297, 309, 315, 327, 333, 345, 351, 363, and 369, and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 4, 10, 22, 28, 40, 46, 58, 64, 75, 82, 94, 100, 112, 118, 130, 136 , 148, 154, 166, 172, 184, 190, 202, 208, 220, 226, 238, 244, 256, 262, 274, 280, 292, 298, 310, 316, 328, 334, 346, 352, 364 , and 370, and an antibody variable light chain sequence having a CDR1 sequence region selected from the group consisting of SEQ ID NOs: 5, 11.23, 29, 41, 47, 59, 65, 77, 83 , 95, 101, 113, 119, 131, 137, 149, 155, 167, 173, 185, 191, 203, 209, 221, 227, 239, 245, 257, 263, 275, 281, 293, 299, 311,317 , 329, 335, 347, 353, 365, and 371, a sequence of CDR2 selected from the group consisting of SEQ ID NOs: 6, 12, 2 4, 30, 42, 48, 60, 66, 78, 84, 96, 102, 114, 120, 132, 138, 150, 156, 168, 174, 186, 192, 204, 210, 222, 228, 240, 246, 258, 264, 276, 282, 294, 300, 312, 318, 330, 336, 348, 354, 366, and 372, and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 7, 13, 25, 31.43, 49, 61.67, 79, 85, 97, 103, 115, 121, 133, 139, 151, 157, 169, 175, 187, 193, 205, 211, 223, 229, 241, 247, 259, 265, 277, 283, 295, 301, 313, 319, 331, 337, 349, 355, 367, and 373; altering at least one amino acid residue within the variable region heavy chain sequence of the antibody region and / or the antibody light chain sequence of the variable region to create at least one altered antibody sequence, and express the altered antibody sequence, as a altered protein.The antibody sequence can also be prepared by antibody screening libraries that have the fixed or minimum essential CDR3 sequences determining binding as described in US20050255552 and diversity in the CDR1 and CDR2 sequences. Screening can be performed according to any suitable screening technology for antibodies screening antibody libraries, such as phage display technology.
[0409] [00409] Molecular biology techniques can be used to prepare and express the altered antibody sequence. The antibody encoded by the altered antibody (s) sequence is one that maintains one, some or all of the functional properties of the HER3-binding antibodies in the present invention described, which functional properties include, but are not limited to , which specifically binds to human HER3 and / or cinomologists, the antibody binds to HER3 and neutralizes HER3 biological activity by inhibiting HER signaling activity in a phospho-HER assay.
[0410] [00410] The functional properties of the altered antibodies can be evaluated using standard assays available in the art and / or described in the present invention, such as those described in the examples (for example, ELISA).
[0411] [00411] In certain embodiments of the antibody engineering methods of the present invention, mutations can be introduced randomly over all or selective or part of a HER3-binding antibody sequence encoding and the resulting HER-binding antibodies can be screened for binding activity and / or other functional properties as described in the present invention. Mutational methods have been described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for creating and tracking antibody mutations using saturation muetiquetaesis, synthetic binding monetiquetaem, or a combination thereof. Alternatively, PCT publication WO 03/074679 by Lazar et al. describes the methods for using computational screening methods to optimize the physicochemical properties of antibodies. Characterization of the Antibodies of the Present Invention
[0412] [00412] The antibodies of the present invention can be characterized by several functional assays. For example, they can be characterized by their ability to neutralize biological activity, inhibiting HER signaling in a phospho-HER assay as in the present invention described, their affinity for the HER3 protein (for example, human HER3 and / or cinomologists ), the binning epitope, its resistance to proteolysis, and its ability to block HER3 downstream signaling. Several methods can be used to measure HER3-mediated signaling. For example, the HER signaling pathway can be monitored by (i) measuring phosphor-HER3, (ii) measuring downstream HER3 phosphorylation or other signaling proteins (eg Akt), (iii) ligand assays blocking as described in the present invention, (iv) heterodimer formation, (v) HER3 gene-dependent expression signature, (vi) receptor internalization, and (vii) HER3 motor cell phenotypes (eg proliferation) .
[0413] [00413] The ability of an antibody to bind to HER3 can be detected by labeling the antibody of interest directly, or the antibody can be unlabeled and binding detected indirectly using various sandwich assay formats known in the art.
[0414] [00414] In some embodiments, the HER3 binding antibodies of the inventive block compete with the binding of a reference HER3 binding antibody to a HER3 polypeptide or protein. These can be completely human, HER3 binding antibodies described above. They may also be other chimeric or humanized, mouse HER3 antibodies that bind to the same epitope as the reference antibody. The ability to block or compete with the binding of the reference antibody indicates that an HER3 binding antibody under test binds to the same or similar epitope as that defined by the reference antibody, or to an epitope that is sufficiently close to the epitope bound by the antibody binding framework HER3. Such antibodies are especially likely to share the beneficial properties identified for the reference antibody. The ability to block or compete with the reference antibody can be determined by, for example, a competition binding assay. With a competition binding assay, the antibody to be tested is examined for the ability to inhibit specific binding of the antibody to a common reference antigen, such as a HER3 polypeptide or protein. A test antibody competes with the reference antibody for specific antigen binding if an excess of the test antibody substantially inhibits binding of the reference antibody. Substantial inhibition means that the test antibody reduces the specific binding of the reference antibody generally by at least 10%, 25%, 50%, 75%, or 90%.
[0415] [00415] There are a number of known binding competition assays that can be used to assess the competition of an HER3 binding antibody with the reference HER3 binding antibody for binding to an HER3 protein. These include, for example, direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al, (1983) Methods in Enzymology 9: 242 a 253.); direct phase assay Solid biotin-avidin EIA (see Kirkland et al, (1986) J. Immunol 137: 3614-3619 ..); direct marked solid phase assay, in solid phase, marked direct sandwich assay (see Harlow and Lane, supra); direct solid phase of RIA tag using I-125 tag (see Morel et al, (1988) Molec Immunol 25: 7 to 15); . direct solid phase biotin-avidin EIA (Cheung et al, (1990) Virology 176: 546 to 552), and direct RIA tags (Moldenhauer et al, (1990.) Scand J. Immunol 32: 77 to 82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells having one, an unlabeled HER3 antibody test and a labeled reference antibody. Competitive inhibition is measured by determining the amount of marker attached to the solid surface or cells in the presence of the test antibody. Normally, the test antibody is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for spatial impairment to occur.
[0416] [00416] To determine whether selected monoclonal HER3 binding antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (eg, Pierce, Rockford, IL reagents). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using a polypeptide from coated HER3-ELISA plates. Biotinylated binding MAb can be detected with an alkaline strepavidin phosphatase probe. To determine the isotype of a purified HER3 binding antibody, isotype ELISA tests can be performed. For example, microtiter plate wells can be coated with 1 µg / ml of anti-human IgG overnight at 4 ° C. After blocking with 1% BSA, the plates are reacted with 1 µg / ml or less of the HER3 binding monoclonal antibody or purified isotypic controls, at room temperature for one to two hours. The wells can then be reacted with either human IgG1 or specific human IgM conjugated to alkaline phosphatase probes. The plates are then developed and analyzed so that the isotype of the purified antibody can be determined.
[0417] [00417] To demonstrate the binding of monoclonal antibodies binding to HER3 antibodies to living cells that express an HER3 polypeptide, flow cytometry can be used. Briefly, cell lines expressing HER3 (grown under normal growing conditions) can be mixed with various concentrations of an HER3 binding antibody in PBS containing 0.1% BSA and 10% fetal calf serum, and incubated at 4 ° C for 1 hour. After washing, the cells are reacted with anti-human IgG fluorescein-labeled antibody under the same conditions as the staining of the primary antibody. The samples can be analyzed by a FACScan instrument using the light and one-sided dispersion properties for the gate in individual cells. An alternative assay using fluorescence microscopy can be used (in addition to or instead of) the flow cytometry assay. The cells can be stained exactly as described above and examined using fluorescence microscopy. This method allows the visualization of individual cells, but the sensitivity may have decreased, depending on the density of the antigen.
[0418] [00418] The HER3-binding antibodies of the present invention can be further tested for reactivity with an antigenic HER3 polypeptide fragment or by Western blotting. Briefly, purified HER3 polypeptides or fusion proteins, or cell extracts from cells expressing HER3, can be prepared and electrophoresed on polyacrylamide dodecyl sulfate gel. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10% fetal calf serum, and probed with the monoclonal antibodies to be tested. Binding human IgG can be detected using anti-human alkaline phosphatase anti-IgG antibody and developed with substrate BCIP / NBT tablets (Sigma Chem. Co., St. Louis, MO).
[0419] [00419] A series of readings can be used to assess the efficacy and specificity of HER3 Antibodies in cell-based assays induced by the heterodimer-forming ligand. The activity can be evaluated using one or more of the following characteristics:
[0420] [00420] (I) Inhibition of HER2 ligand-induced heterodimerization with other members of the EGF family of a target cell line, for example, MCF-7 breast cancer cells. Immunoprecipitation of HER2 cell lysate complexes can be performed with an antibody to the specific receptor, and the absence / presence of other EGF receptors and their biologically relevant ligands within the complex can be analyzed after electrophoresis / Western blotting by means of from probing with antibodies to the other EGF receptors.
[0421] [00421] (Ii) inhibition of the activation of signaling pathways by means of the heterodimers activated by the ligands. Association with HER3 appears to be key for other members of the EGF receptor family to elicit maximum cellular response after ligand binding. In the case of a deficient HER3 kinase, HER2 provides a functional tyrosine kinase domain to allow signaling to occur after binding of the growth factor ligands. In this way, cells that co-express HER2 and HER3 can be treated with the ligand, for example, heregulin, in the absence and presence of inhibitor and the effect on HER3 tyrosine phosphorylation monitored in a number of ways, including immunoprecipitation of treated HER3 cell lysates and subsequent western blotting using anti-phosphotyrosine antibodies (see Agus op. cit. for more details). Alternatively, a high throughput assay can be developed by trapping HER3 solubilized lysates into the wells of a 96-well plate coated with an anti-HER3 receptor antibody, and the level of tyrosine phosphorylation measured using, for example, europium - anti-labeled phosphotyrosine, as defined by Waddleton et al., (2002) Anal. Biochem. 309: 150 to 157.
[0422] [00422] In a broader form of this approach, effector molecules that are known to be activated downstream of activated receptor heterodimers, such as mitogenic activator kinases (MAPK) and Akt, can be analyzed directly, through immunoprecipitation of treated lysates and immunodetection with antibodies that detect the activated forms of these proteins, or by analyzing the ability of these proteins to modify / activate specific substrates.
[0423] [00423] (III) inhibition of cell ligand-induced proliferation. A variety of cell lines are known to co-express combinations of ErbB receptors, for example, breast and prostate cancer cell lines. The assays can be performed in 24/48/96-well formats with the base reading around DNA synthesis (incorporation of tritiated thymidine), increase in the number of cells (staining with crystal violet), etc.
[0424] [00424] A series of readings can be used to evaluate the efficacy and specificity of HER3 Antibodies in assays based on cells forming homo- and heterodimers independent of the ligand. For example, HER2 overexpression triggers independent activation of the kinase domain ligand as a result of spontaneous dimer formation. Overexpression of HER2 generates either homo or heterodimers with other molecules such as HER HER1, HER3 and HER4.
[0425] [00425] Ability of antibodies or fragments thereof to block in vivo growth of tumor xenografts from human tumor cell lines whose tumorigenic phenotype is known to be at least partially dependent on activation by the HER3 ligand, for example , BxPC3 heterodimer signaling cells, pancreatic cancer cells etc. This can be evaluated in immunocompromised mice, either alone or in combination with a cytotoxic agent appropriate for the cell line in question. Examples of functional tests are also described in the Example section below. Prophylactic and therapeutic uses
[0426] [00426] The present invention provides methods of treating a disease or disorder associated with the HER3 signaling pathway by administration to a subject who needs an effective amount of the antibodies of the present invention. In a specific embodiment, the present invention provides a method of treating or preventing cancers (for example, breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, cancer prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear soft tissue sarcoma, malignant mesothelioma, neurofibromatosis, kidney cancer and melanoma), by administering it to a subject who needs an effective amount of the antibodies of the present invention. In some embodiments, the present invention provides methods of treating or preventing cancers associated with an HER signaling pathway, by administration to a subject who needs an effective amount of the antibodies of the present invention.
[0427] [00427] In a specific embodiment, the present invention provides methods of treating cancers associated with an HER signaling pathway that include, but are not limited to breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath Schwannoma tumors, head and neck, bladder cancer, esophageal cancer, glioblastoma, clear cell soft tissue sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, and melanoma.
[0428] [00428] HER3 antibodies can also be used to treat or prevent other disorders associated with aberrant or deficient HER signaling, including, but not limited to, respiratory diseases, osteoporosis, osteoarthritis, polycystic kidney disease, diabetes, schizophrenia, vascular disease, disease cardiac, non-oncogenic proliferative diseases, fibrosis, and neurodegenerative diseases such as Alzheimer's disease.
[0429] [00429] Suitable agents for the treatment of combination with HER3-binding antibodies include standard assisting agents known in the art that are capable of modulating the ErbB signaling pathway. Suitable examples of standard HER2 care agents include, but are not limited to, Herceptin and Tykerb. Suitable examples of standard care agents for EGFR include, but are not limited to, Iressa, Tarceva, Erbitux and Vectibix. Other agents that may be suitable for combination treatment with HER3-binding antibodies include, but are not limited to, those that modulate tyrosine kinase receptors, protein G of coupled receptors, growth / survival signal transduction pathways, receptors nuclear hormones, apoptotic pathways, cell cycle and angiogenesis. Diagnostic Uses
[0430] [00430] In one aspect, the present invention includes diagnostic tests for the determination of HER3 protein and / or nucleic acid expression, as well as the function of the HER3 protein, in the context of a biological sample (e.g., blood, serum , cells, tissues) or an individual afflicted with cancer, or is at risk of developing cancer.
[0431] [00431] Diagnostic assays, such as competitive assays are based on the ability of a labeled analogue (the "marker") to compete with the test sample analyte for a limited number of binding sites on a common binding partner. The liaison partner is generally insolubilized before or after competition and then the tracer and analyte linked to the liaison partner are separated from the tracer and unbound analyte. This separation is carried out by decanting (when the bonding partner has been pre-solubilized) or by centrifuging (when the bonding partner has been precipitated after the competitive reaction). The amount of test sample analyte is inversely proportional to the amount of tracer bound as measured by the amount of marker substance. Dose-response curves with known amounts of analyte are prepared and compared with the test results in order to quantitatively determine the amount of analyte present in the test sample. These assays are called ELISA systems when enzymes are used as detectable markers. In such an assay, the competitive binding between antibodies and the HER3 antibody binding results in the HER3 protein, preferably the HER3 epitopes of the present invention, is a measure of the antibodies in the serum sample, more particularly, neutralizing antibodies in the serum sample.
[0432] [00432] A significant vanet of the assay is that the measurement is made directly from neutralizing antibodies (ie, those that interfere with the binding of the HER3 protein, specifically, the epitopes). Such an assay, in particular in the form of an ELISA test, has considerable applications in the clinical setting and in routine blood testing.
[0433] [00433] Another aspect of the present invention provides methods for determining the expression of HER3 nucleic acid or HER3 protein activity in an individual, to thereby select the appropriate therapeutic or prophylactic agents for that individual (in the present invention referred to as "pharmacogenesis" -nomics "). Pharmacogenomics allows the selection of agents (for example, drugs) for the therapeutic or prophylactic treatment of an individual based on the individual's genotype (for example, the examined individual's genotype to determine the individual's ability to respond to a particular agent.)
[0434] [00434] Yet another aspect of the present invention relates to monitoring the influence of agents (e.g., drugs) on the expression or activity of HER3 protein in clinical trials. Pharmaceutical compositions
[0435] [00435] To prepare pharmaceutical or sterile compositions that include an HER3 binding antibody of intact fragments (or binding), HER3 binding antibodies (intact or binding fragments) are mixed with a pharmaceutically acceptable carrier or excipient. The compositions may additionally contain one or more other therapeutic agents that are suitable for the treatment or prevention of cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath Schwannoma tumors, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, clear soft tissue sarcoma, malignant mesothelioma, neurofibromatosis, kidney cancer and melanoma).
[0436] [00436] Therapeutic and diagnostic agent formulations can be prepared by mixing with physiologically acceptable vehicles, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions, suspensions or ( see, for example, Hardman et al., (2001) Goodman and Gilman, The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Pratice of Pharmacy, Lippincott, Williams and Wilkins , New York, NY; Avis, et al. (Eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al (eds ...) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie Excipient Toxicity (2000) and safety, Marcel Dekker, Inc., New York, NY) .
[0437] [00437] Selection of a therapeutic agent administration regimen depends on several factors, including the rate of rotation of the entity's serum or tissue, the level of symptoms, the immunogenicity of the entity and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapy to the patient consistent with an acceptable level of side effects. Therefore, the amount of biological product delivered depends in part on the particular entity and the severity of the condition being treated. Guidance on selecting appropriate doses of antibodies, cytokines, and small molecules is available (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;. Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibody and Peptide Therapy, in Autoimmune Diseases, Maree Dekker, New York, NY ,. Baert et al, (2003) New Engl J. Med. 348: 60 to 608; Milgrom et al, (1999) New Engl J. Med. 341: 1966 to 1973, Slamon et al, (2001) New Engl J. Med. 344: 783 to 792; Beniaminovitz et al, (2000) New Engl J. Med. 342: 613 to 619;. Ghosh et al, (2003) New Engl J. Med. 348: 24-32, Lipsky et al, (2000) New Engl.. J Chem. 343: 1594 to 1602).
[0438] [00438] The determination of the appropriate dose is made by the clinician, for example, using the parameters or factors known or suspected in the art, to affect the treatment or predicted to affect the treatment. Generally, dosing starts with a little less than the optimum dose and is increased by small increments until after that the desired or optimum effect is achieved in relation to any negative side effects. Important diagnostic measures include those of the symptoms of, for example, inflammation or the level of inflammatory cytokines produced.
[0439] The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of factors, including the pharmacokinetic activity of the particular compositions of the present invention used, or their ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the compound particular to be used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and previous medical history of the patient to be treated, and how factors known in the medical arts.
[0440] [00440] Compositions comprising the antibodies or fragments thereof of the present invention may be provided by means of continuous infusion or by means of doses at intervals of, for example, one day, one week, or 1 to 7 times a week . Doses can be delivered intravenously, subcutaneously, topically, orally, nasal, rectally, intramuscularly, intracerebrally or by inhalation. A specific dose protocol is one that involves the dose or frequency of the maximum dose that avoids significant undesirable side effects. A total weekly dose can be at least 0.05 μg / kg of body weight, at least 0.2 μg / kg, at least 0.5 pg / kg, at least 1 μg / kg, at least 10 μg / kg, at least 100 μg / kg, of at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 25 mg / kg, or at least 50 mg / kg (see, for example, Yang et al, (2003) New Engl J. Med. 349: 427-434, Herold et al, (2002) New Engl J. Med. 346: 1692 to 1698, Liu et al, (1999) J. Neurol Neurosurg Psych 67: 451 to 456; Portielji et al, (2003) Cancer Immunol Immunother 52: 133 to 144). The desired dose of antibodies or fragments thereof is approximately the same as for an antibody or polypeptide, on a mole / kg basis weight basis. The desired plasma concentration of the antibodies or their fragments is approximately one moles / kg of base body weight. The dose can be at least 15 g of at least 20 pg, at least 25 pg, at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg at least 55 pg, at least 60 pg, at least 65 pg, at least 70 pg, at least 75 pg, at least 80 pg, at least 85 pg, at least 90 pg, in at least 95 pg, or at least 100 pg. The doses administered to a subject can list at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.
[0441] [00441] For antibodies or fragments thereof of the present invention, the dosage administered to a patient can be from 0.0001 mg / kg to 100 mg / kg of the patient's body weight. The dosage can be between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg at 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg,, 01 to 0.25 mg / kg or 0.01 to 0, 10 mg / kg of the patient's body weight.
[0442] [00442] The dosage of antibodies or fragments thereof of the present invention can be calculated using the patient's weight in kilograms (kg), multiplied by the dose to be administered in mg / kg. The dosage of antibodies or fragments thereof of the present invention can be 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 pg / kg or less, 80 μg / kg or less, 75 μg / kg or less, 70 μg / kg or less, 65 μg / kg or less, 60 μg / kg or less, 55 μg / kg or less, 50 μg / kg or less, 45 μg / kg or less, 40 μg / kg or less, 35 μg / kg or less, 30 μg / kg or less, 25 μg / kg or less, 20 μg / kg or less, 15 μg / kg or less, 10 μg / kg or less, 5 μg / kg or less, 2.5 pg / kg or less, 2 μg / kg or less, 1.5 μg / kg or less, 1 μg / kg or less , 0.5 μg / kg or less, or 0.5 μg / kg or less of the patient's body weight.
[0443] [00443] Unit dose of the antibodies or fragments thereof of the present invention can be from 0.1 mg to 20 mg, 0.1 mg and 15 mg, 0.1 mg and 12 mg, 0.1 mg and 10 mg, 0.1 mg and 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0, 25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg , 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
[0444] [00444] The dosage of antibodies or fragments thereof of the present invention can reach a serum titer of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least , 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml, or at least at least 400 μg / ml in a subject. Alternatively, the dosage of antibodies or fragments thereof of the present invention can achieve a serum titer of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 pg / ml, at least 15 μg / ml, at least 20 ng / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml, or at least 400 μg / ml in the subject.
[0445] [00445] The doses of antibodies or fragments thereof of the present invention can be repeated and the administrations can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days , 2 months, 75 days, 3 months, or at least 6 months.
[0446] [00446] An effective amount for a particular patient can vary depending on factors such as the condition being treated, the patient's general health, the route and dose of method of administration and the severity of side effects (see, for example, Maynard et al., (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla .; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0447] [00447] The route of administration can be through, for example, injection, application, topical or cutaneous, or by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by systems of controlled or implant release (see, for example, Sidman et al., (1983) Biopolymers 22: 547-556; Langer et al., (1981) J. Biomed. Mater. Res. 15: 167 to 277; Langer ( 1982) Chem. Tech. 12: 98 to 105; Epstein et al., (1985) Proc. Natl. Acad. Sci. USA 82: 3688 to 3692; Hwang et al., (1980) Proc. Natl. Acad. Sci USA 77: 4030 to 4034; US Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. In addition, pulmonary administration can also be employed, for example, by the use of an inhaler or nebulizer, and formulation with an aerosol-forming agent. See, for example, US Patent No. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and PCT Publication No. WO 92 / 19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety.
[0448] [00448] The composition of the present invention can also be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be understood by the person skilled in the art, the route and / or mode of administration will vary depending on the desired results. Selected routes of administration for the antibodies or fragments of thatof the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent different modes of administration than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtraqueal, subcutaneous, subcuticular, injection, intra-articular, subcapsular, subarachnoid, intra epidural and intrasternal injection and infusion. Alternatively, a composition of the present invention can be administered via a non-parenteral route, such as a topical, epidermal or mucous route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one embodiment, the antibodies or fragments thereof of the present invention are administered by means of the infusion. In another embodiment, the multispecific epitope-binding protein of the present invention is administered subcutaneously.
[0449] [00449] If the antibodies or fragments thereof of the present invention are administered from a controlled release or a controlled release system, a pump can be used to achieve a controlled or sustained release (see, Langer, supra; Sefton (1987) CRC Crit Ref Biomed Eng. 14: 20; Buchwald et al, (1980), Surgery 88: 507, Saudek et al, (1989) N. Engl J. Med. 321: 574). Polymeric materials can be used to achieve a controlled or sustained release of the therapies of the present invention (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida. (1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also, Levy et al., (1985) Science 228: 190; During et al., (1989) Ann. Neurol. 25: 351; Howard et al., (1989) J. Neurosurg. 71: 105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154, and PCT Publication No. WO 99 / 20253. Examples of polymers used in controlled release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate) ), poly (acrylic acid), poly (ethylene-vinyl acetate), poly (methacrylic acid), polyglycolides (PLG), polyanhydrides, poly (N-vinyl-pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylates (PLA), poly (bark-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable in storage, sterile, and biodegradable. A controlled or sustained release system can be placed in close proximity to the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp 115 a 138 (1984)).
[0450] [00450] Controlled release systems are discussed in the review by Langer, (1990), Science 249: 1527 to 1533). Any technique known to one skilled in the art can be used to produce controlled release formulations comprising one or more antibodies or fragments thereof. See, for example, US Patent No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., (1996), Radiotherapy & Oncology 39: 179-189, Song et al. , (1995) Journal of Science PDA Pharmaceutical & Technology 50: 372-397, Cleek et al., (1997) Pro. Int'l. Symp. To control. Bioact report Mater. 24: 853 to 854, and Lam et al., (1997) Proc. Int'l. Symp. Control Bioact Report. Mater. 24: 759 to 760, each of which is incorporated herein by reference in its entirety.
[0451] [00451] If the antibodies or fragments thereof of the present invention are administered topically, they can be formulated in the form of an ointment, cream, transdermal plaster, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or any other form well known to one skilled in the art. See, for example, Remington’s Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, ed 19., Pub Mack. Co., Easton, Pa. (1995). For topical non-sprayable dosage forms, viscous, semi-solid or solid forms comprise a vehicle or one or more excipients compatible with topical application and having a dynamic viscosity, in some cases, greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, and the like, which are, if desired, sterilized or mixed with auxiliary agents (for example, preservatives, stabilizers, agents wetting agents, buffers, or their salts) to influence various properties, such as, for example, osmotic pressure. Other suitable dosage forms include topical spray aerosol preparations, in which the active ingredient, in some cases, in combination with a solid or liquid inert carrier, is packaged in a mixture with a volatile pressurized (for example, a gaseous propellant such as like freon) or a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art.
[0452] [00452] If the compositions comprising the antibodies or fragments thereof are administered intranasally, it can be formulated in an aerosol, spray, mist or droplet form. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently administered in the form of an aerosol spray presentation from pressurized packages or a nebulizer, with the use of a suitable propellant (for example, dichlorodifluoromethane , trichlorofluoromethane, carbon, dichlorotetrafluoro-roethane dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, for example, gelatin) for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.
[0453] [00453] Methods for coadministration or treatment with a second therapeutic agent, for example, a cytokine, steroid chemotherapeutic agent, antibiotics, or radiation, are known in the art (see, for example, Hardman et al., ( Eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach , Lippincott, Williams & Wilkins, Phila., Pa .; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of the therapy can decrease symptoms by at least 10%; by at least 20%, at least about 30%, at least 40%, or at least 50%.
[0454] [00454] Additional therapies (for example, prophylactic or therapeutic agents), which can be administered in combination with the antibodies or fragments thereof of the present invention can be administered less than 5 minutes apart, less than 30 minutes apart , 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart interval, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, in about 6 hours to about 7 hours apart, about 7 hours and about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, in about 11 hours around 12 hours apart, about 12 hours to 18 hours apart, 18 hours and 24 hours apart o, 24 hours and 36 hours apart, 36 hours and 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours and 96 hours apart, or 96 hours to 120 hours apart from the antibodies or fragments thereof. The two or more therapies can be administered within a visit by the same patient.
[0455] [00455] The antibodies or fragments thereof of the present invention and the other therapies can be administered cyclically. Cyclic therapy involves the administration of a first therapy (for example, a first prophylactic or therapeutic agent), for a period of time, followed by the administration of a second therapy (for example, a second prophylactic or therapeutic agent), for a period of time, optionally, following the administration of a third-party therapy (for example, a prophylactic or therapeutic agent), for a period of time and so on, and repeating this sequential administration, that is, the cycle of in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and / or to improve the effectiveness of the therapies.
[0456] [00456] In certain embodiments, the antibodies or fragments thereof of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, U.S. Patent No. 4,522,811; 5,374,548 and 5,399,331. Liposomes can comprise one or more units that are selectively transported in specific cells or organs, thereby improving delivery of the target drug (see, for example, Ranade, (1989) J. Clin. Pharmacol. 29: 685). Examples of targeting moieties include folate or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); Mannosids (Umezawa et al, (1988) Biochem Biophys Res Commun 153: 1038), Antibodies (Bloeman et al, (1995) FEBS Lett 357: 140; Owais et al, (1995) Antimicrob Agents Chemother 39: 180); protein receptor (. Briscoe et al, (1995) Am. J. Physiol. 1233: 134), p 120 (Schreier et al, (1994) J. Biol Chem 269: 9090), see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273.
[0457] [00457] The present invention provides protocols for administering antibodies to the pharmaceutical composition comprising fragments thereof of the present invention alone or in combination with other therapies for a subject in need thereof. The therapies (for example, prophylactic or therapeutic agents) of the combination therapies of the present invention can be administered concomitantly or sequentially to a subject. The treatment (for example, prophylactic or therapeutic agents) of the combination therapies of the present invention can also be administered cyclically. Cyclic therapy involves the administration of a first therapy (for example, a first prophylactic or therapeutic agent), for a period of time, followed by the administration of a second therapy (for example, a second prophylactic or therapeutic agent), for a period of time and repeating this sequential administration, ie the cycle, in order to reduce the development of resistance to one of the therapies (for example, agents) to avoid or reduce the side effects of one of the therapies (for example, agents ), and / or to improve the effectiveness of therapies.
[0458] [00458] The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can be administered to a patient at the same time. The term "concurrently" is not limited to the administration of therapies (for example, prophylactic or therapeutic agents) at exactly the same time, but instead, it is understood that a pharmaceutical composition comprising the antibodies or fragments thereof of the present invention are administered to a subject with a sequence and within a period of time in such a way that the antibodies of the present invention can act in conjunction with the therapy of other (s) to provide a greater benefit than if they were administered in another form. For example, each therapy can be administered to an individual at the same time or sequentially in any order at different points in time, however, if not administered at the same time, they must be administered sufficiently close in time to provide the desired effect. therapeutic or prophylactic. Each therapy can be administered to an individual separately, in any suitable form and by any suitable route. In various modalities, therapies (for example, prophylactic or therapeutic agents) are administered to a patient less than 15 minutes, less than 30 minutes, less than 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, in about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart , about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hour break, or 1 week. In another embodiment, two or more therapies (for example, prophylactic or therapeutic agents) are administered to a patient on the same visit.
[0459] [00459] The prophylactic or therapeutic agents of the combination therapies can be administered to a subject with the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered simultaneously to a subject in separate pharmaceutical compositions. Prophylactic or therapeutic agents can be administered to a patient by the same or different routes of administration.
[0460] [00460] The present invention having been fully described, is further illustrated by the following examples and claims, which are illustrative and are not intended to be limited further. EXAMPLES Example 1: Methods, materials and screening for antibodies (I) Cell lines
[0461] [00461] BXPC-3, SK-BR-3, BT-474, MDA-MB-453, FADU and MCF-7 cell lines were acquired from ATCC and routinely maintained in growth medium supplemented with 10% serum fetal bovine (FBS). (Ii) Generation of HER3 Human, Cino, Mouse and Recombinant Rat Vectors
[0462] The murine HER3 extracellular domain was amplified by PCR from mouse brain cDNA (Clontech) and the sequence verified by comparison with RefSeq NM_010153. Mouse ECD HER3 was reverse transcribed from cellular Rat-2 mRNA and the sequence verified by comparison with NM_017218. The HER3 cynomologist cDNA model was generated using RNA from various tissues (cyno Zyagen Laboratories), and the RT-PCR product cloned into pCR-TOPO ®-XL (Invitrogen) before sequencing both strands. Human HER3 was derived from a human fetal brain cDNA library (source) and the sequence verified by comparison with NM_001982.
[0463] [00463] To generate labeled recombinant proteins, human HER3, cino, mouse and rat was amplified by PCR using Taq Pwo polymerase (Roche Diagnostics). The PCR amplified products were gel purified and cloned into a pDonR201 (Invitrogen) gateway input vector that had previously been modified to include a frame at the CD33 command sequence N terminal and a C terminal LABEL, for example, LABEL FLAG. The LABEL allows the purification of monomeric proteins using a monoclonal anti-LABEL antibody. The target genes were flanked with attB1 and attB2 allowing recombination in vectors adapted to the destination gateway property (for example, pcDNA3.1), using the Gateway ® cloning technology (Invitrogen). The recombination reactions were performed using a Gateway LR reaction with proprietary target vectors containing a CMV promoter to create the LABEL expression vectors, although any commercially available vector can be used.
[0464] [00464] In addition the recombinant proteins were generated HER3 which fused the HER3 ECD upstream of a Factor X cleavage site C terminal and the human IgG hinge and the Fc domain to create an Fc-tag protein. To achieve this goal, the PCR was the various HER3 ECD's amplified and cloned into a vector (for example, pcDNA3.1) modified to contain an internal Factor X Terminal C fusion structure from the HFC- hinge location. The open reading frame was generated flanked with the attB1 and attB2 sites for additional cloning with Gateway ® recombinant cloning technology (Invitrogen). A Gateway LR reaction was used to transfer HER3-Fc to a target construct expression containing a CMV promoter. The expression constructs of HER3 mutations were generated using standard protocols of the targeted muetiquetaênese site and the resulting vector sequence verified. Table 8. Generation of HER3 expression vectors. Amino acid numbering HER3 is based on NP_001973 (human), NP_034283 (mouse) and NP_058914 (rat).
[0465] [00465] The desired HER3 recombinant proteins were expressed in derived HEK293 cell lines previously adapted for suspension culture and cultured in a serum-free medium, proprietary Novartis. Small scale expression verification was performed in transient assays of 6 wells of transfection plate as a function of lipofection. Large-scale protein production through transient transfection was performed at 10 to 20 scale L of the Wave ™ bioreactor system (Wave Biotech). Polyethyleneimine DNA (Polysciences) was used as a carrier plasmid, in the proportion of 1: 3 (w: w). Cell culture supernatants were harvested 7 to 10 days after transfection and concentrated by cross-flow filtration and diafiltration prior to purification. (Iv) Purification of labeled Protein
[0466] [00466] The labeled recombinant HER3 proteins (e.g., LABEL-HER3) were purified by collecting the cell culture supernatant and concentrating 10 times by cross-flow filtration with a 10 kDa cut from the filter (Fresenius). An anti-LABEL column was prepared by coupling an activated anti-CNBR Sepharose 4B monoclonal antibody activated with a final ratio of 10 mg of antibody per ml of resin. The concentrated supernatant was applied to a 35ml anti-Label column, with a flow rate of 1 to 2 ml / minute. After the PBS washing baseline, the bound material was eluted with 100 mM glycine (pH 2.7), neutralized and sterilized by filtration. Protein concentrations were determined by measuring the absorbance at 280 nm and using a theoretical conversion factor of 0.66 AU / mg. The purified protein was finally characterized by SDS-PAGE, N-terminal sequencing and LC-MS. (V) Purification Label Fc
[0467] [00467] The concentrated cell culture supernatant was applied to a 50 ml protein A Sepharose Fast Flow column with a flow rate of 1 ml / min. After washing with baseline PBS, the column was washed with 10 column volumes of 10 mM NaH2PO4 / 30% (v / v) isopropanol, pH 7.3 followed by 5 column volumes of PBS. Finally, the bound material was eluted with 50 mM Citrate / 140 mM NaCl (pH 2.7), neutralized and sterilized by filtration. (Vi) Aeration of cell overexpression lines
[0468] [00468] To generate a cell line that expresses high levels of HER3 on the cell surface, a mammalian expression vector was constructed containing an insert encoding a leading CD33 sequence upstream of amino acid residues 20 to 667 of HER3 human cast in frame for the 669 to 1210 amino acid residues of human EGFR. When expressed in mammalian cells, the resulting chimeric protein contains an N-terminal of the extracellular HER3 and transmembrane domain and a C-terminal domain of the cytoplasmic EGFR. The HER3 / 1 vector was transfected in CHO-S cells (Invitrogen) and stable pools generated after antibiotic selection. The resulting cell line (CHO HER3 / 1) expressed the high levels of HER3 extracellular domain on the cell surface. (Vii) Pannings of GOLD ® HuCAL
[0469] [00469] For the selection of antibodies that recognize multiple human HER3, panning strategies were employed. The therapeutic antibodies against human HER3 protein were generated through the selection of clones that have high binding affinities, using the commercially available phage display library, the MorphoSys OURO HuCAL ® library, as the source of the antibody. The phagemid library is based on the HuCAL ® concept (Knappik et al., (2000) J Mol Biol 296: 57 to 86) and employs CysDisplay ® technology to display Fab on the phage surface (WO01 / 05950 for Lohning).
[0470] [00470] For the isolation of standard anti-HER3 antibodies, as well as RapMAT, panning strategies were performed using solid phase, solution, and whole cell differential whole cell panning approaches. (Viii) Panning in Solid Phase
[0471] [00471] To identify anti-HER3 antibodies, a variety of solid-phase panning strategies were performed using different HER3 recombinant proteins. To perform each round of panning in solid phase, Maxisorp plates (Nunc) were coated with the HER3 protein. Proteins labeled or captured using plates previously coated with anti-Fc antibody (goat or mouse anti-human IgG, Jackson Immuno Research), anti-Tag or antibody via passive adsorption. The coated plates were washed with PBS and blocked. The coated plates were washed twice with PBS before adding the OURO ® HuCAL- antibody phage for 2 hours at room temperature on a shaker. Bound phage were eluted, added to E. coli TG-1 and incubated during phage infection. Subsequently, the infected bacteria were isolated and plated on agar plates. The colonies were scraped from the plates and the phages were rescued and amplified. Each panoramic HER3 strategy contained individual panning rounds and contained unique antigens, antigen concentrations and washing rigor. (Ix) Panning Solution Phase
[0472] [00472] Each round of panning solution phase was performed using several biotinylated recombinant HER3 proteins in the presence or absence of neuregulin 1-β1 (R & D Systems). The proteins were biotinylated using the EZ-link Sulfo-NHS-LC biotinylation kit (Pierce) according to the manufacturer's instructions. 800μΙ of streptavidin-bound magnetic beads (Dynabeads, Dynal) were washed once with PBS and blocked overnight with Chemiblocker (Chemicon). OURO® HuCAL antibodies on phages and the appropriate biotinylated HER3 were incubated in a reaction tube. The streptavidin magnetic beads were added over 20 minutes and were collected with a magnetic particle separator (Dynal). Bound phage were eluted from the Dynabeads by adding DTT buffer containing to each tube and adding E. coli TG-1. The phage infection was performed in the same way as described for solid phase panning. Each panoramic HER3 strategy contained the individual panning rounds and contained unique antigens, the antigen concentrations and washing rigor. (X) Cell based panning
[0473] [00473] For cell panning, the GOLD HuCAL ® phage antibodies were incubated with about 107 cells in a rotator for 2 hours at room temperature, followed by centrifugation. The pellet of cells was isolated on phage that were eluted from the cells. The supernatant was collected and added to the E. coli TG-1 culture continued by the process described above. Two cell-based strategies were used to identify anti-HER3 antibodies:
[0474] [00474] a) whole cell panning: In this strategy a variety of intact cell lines were used as antigens.
[0475] [00475] b) panning of whole cells Differentials: This strategy consisted sequentially of the cell antigens and recombinant proteins HER3 (see 1981.09 as an example). Cell-based pannings were performed as described above whereas solid-phase panning protocols were used when recombinant proteins were used as antigens. The washes were performed using PBS (2-3X) and PBST (2-3X). (Xi) Generation of RapMAT ™ library and pannings
[0476] [00476] In order to increase the binding affinity of the antibody while maintaining library diversity, the second round of both solution and solid phase pannings were introduced in the RapMAT ™ process while the third round of panning strategies of whole cell and differential whole cell were inserted (Prassler et al., Immunotherapy (2009), 1: 571 to 583. RapMAT ™ libraries were generated by subcloning Fab encoding phage inserts selected by panning on the pMORPH vector display ® 25_bla_LHCand were digested to generate H-CDR2 RapMAT ™ libraries and L-CDR3 RapMAT ™ libraries using specific restriction enzymes. The inserts were replaced by maturation cassettes (TRIM Virnekas et al, (1994) Nucleic Acids Research 22: 5600 to 5607 ) of H-CDR2 or L-CDR3 according to the pool of the composition sizes. The libraries were estimated between 8x106-1x108 clones. RapMAT antibody-phage were produced and subjected to two additional cycles of solid phase solution, or panning cells using the experimental methods described above. Example 2: Transient expression of anti-HER3 IgG
[0477] [00477] Adapted HEK293-6E cell suspension was grown in a BioWave20 at a density of approximately 2 x 106 viable cells / mL. The cells were transiently transfected with the relevant sterile DNA: PEI-MIX and still cultured. Seven days after the transfection, the cells were removed by means of cross-flow filtration using 0.2 µm filters (Fresenius). The cell-free material was concentrated with cross-flow filtration using a cut off 10 kDa filter (Fresenius) and the concentrate was sterilized by means of filtration through a stericup filter (0.22 nm). The supernatant was sterilized and stored at 4 ° C. Example 3: Purification of anti-HER3 IgG antibodies
[0478] [00478] IgG purification was performed in explorer 100 Akta Air chromatography system at 6 ° C in a cooling cabinet, using an XK16 / 20 column with 25 mL of self-packed Sure MabSelect resin (all GE Healthcare ). All flow rates were 3.5 mL / min, except for the load, at a pressure limit of 5 bar. The column was equilibrated with three column volumes of PBS before loading the filtered fermentation supernatant at 2.0 ml / min. The column was washed with 8 column volumes of PBS. IgG was eluted with a pH gradient, starting from 50 mM citrate / 70 mM NaCl (pH 4.5), going down linearly in 12 column volumes of 50 mM citrate / 70 mM NaCl ( pH 2.5), followed by a constant volume step of column 2 of the same pH 2.5 buffer. Fractions containing IgG were collected and immediately neutralized and sterilized by filtration (Millipore Steriflip, 0.22 µm). OD280 was measured and the protein concentration calculated based on the sequence data. The pools were tested separately for aggregation (SEC-MALS) and purity (SDS-PAGE and MS). Example 4: Expression and Purification of Fab-HuCAL ® antibodies in E. coli
[0479] [00479] Expression of Fab fragments encoded by pMORPH ® X9_Fab_MH in TG-1 cells was performed in shake flask cultures with 500 ml of YT medium supplemented with 2x 34 ug / ml chloramphenicol. The cultures were stirred at 30 ° C until OD600nm reached 0.5. Expression was induced by the addition of 0.75 mM IPTG (isopropyl-beta-D-thiogalactopyranoside), for 20 hours at 30 ° C. The cells were burst using lysozyme. The Fab His6-tag fragments were isolated using IMAC (Bio-Rad). Dulbecco's 1x PBS exchange buffer (pH 7.2) was performed using PD10 columns. The samples were sterilized by means of filtration (0.2 mm). Protein concentrations were determined using UV spectrophotometry. The purity of the samples was analyzed in the denaturant, reducing the SDS-PAGE by 15%. The homogeneity of the Fab preparation was determined in its native state using size exclusion chromatography (HP-SEC) with calibration standards Example 5: Affinity of HER3 antibodies (KD) Measured by Titration of the equilibrium solution (SET)
[0480] [00480] The affinity determination in solution was carried out essentially as previously described (Friguet et al., (1985) J Immunol Methods 77: 305 to 19). In order to improve the sensitivity and accuracy of the SET method, it was transferred from classic ELISA to ECL-based technology (Haenel et al., (2005) Anal Biochem 339: 182 to 84).
[0481] [00481] Unlabeled HER3 tag (human, mouse, mouse or cino) described above was used for the determination of affinity for SET.
[0482] [00482] The data were evaluated with XLfit software (Business Solutions ID) the application of customized monetiquetaem models. For the determination of each IgG KD in the following model it was used (modified according to Piehler, et al (Piehler et al., (1997) J Immunol Methods 201: 189 to 206).
[0483] [00483] [IgG]: applied concentration of total IgG
[0484] [00484] x: total applied soluble antigen concentration (binding sites)
[0485] [00485] Bmax: maximum IgG signal without antigen
[0486] [00486] KD: affinity Example 6: Determination of cell-binding antibodies by FACS
[0487] [00487] The binding of antibodies to the endogenous human antigen expressed in human cancer cells has been evaluated by FACS. In order to determine the EC50 values of antibody, SK-BR-3 cells were harvested with Accutase and diluted to cells 1 x 106 / ml in FACS buffer (PBS / 3% FBS / 0.2% NaN3). 1 x 105 cells / well were added to each well of a 96-well plate (Nunc) and centrifuged at 210 g for 5 minutes at 4 ° C before removal of the supernatant. Serial dilutions of test antibodies (diluted in 1: 4 dilution steps with FACS buffer) were added to the pelleted cells and incubated for 1 hour on ice. The cells were washed and pelleted three times with 100 µL of FACS buffer. Goat anti-human IgG PE (Jackson ImmunoResearch) diluted 1/200 with FACS buffer was added to the cells and incubated on ice for 1 hour. The additional washing steps were performed three times with 100 µL of FACS buffer followed by the 210 g centrifugation steps for 5 minutes at 4 ° C. Finally, the cells were resuspended in 200 µl of FACS buffer and fluorescence values were measured with a FACSArray (BD Biosciences). The amount of cell surface bound to anti-HER3 antibody was assessed by measuring the mean channel fluorescence. Example 7: HER3 domain and mutant link
[0488] [00488] Maxisorp 96-well plates (Nunc) were coated overnight at 4 ° C with 200 ng of the appropriate human recombinant protein (HER3-Tag, D1-2-Tag, D2-Tag, D3-4-Tag, D4-Tag, HER3 K267A-Tag, HER3 L268A-Tag, HER3 K267A / L268A and an irrelevant marked control). All wells were then washed three times with PBS / 0.1% Tween-20, blocked for one hour, with PBS / 1% BSA / 0.1% Tween-20 and washed three times with PBS / 0, 1% Tween-20. HER3 Anti-Antibodies were added to the corresponding wells to a final concentration of 10 ng / ml, the appropriate wells were added and incubated at room temperature for two hours. The plates were washed three times with PBS / 0.1% Tween-20, prior to the addition of the appropriate bound peroxidase detection antibody diluted 1 / 10,000 in PBS / 1% BSA / 0.1% Tween-20 . The detection antibodies used were goat anti-mouse (Pierce, 31432), rabbit anti-goat (Pierce, 31402), and goat anti-human (Pierce, 31412). The plates were incubated at room temperature for one hour before washing three times with PBS / 0.1% Tween-20. 100 µl of TMB (3.3 ', 5.5' tetramethylbenzidine), substrate solution (BioFx) was added to all wells for 6 minutes before stopping the reaction with 50 µl of 2.5% H2SO4. The extent of binding of the HER3 antibody to each recombinant protein was determined by measuring the DO450 using a SpectraMax plate reader (Molecular Devices). Where appropriate, dose response curves were analyzed using Graphpad Prism. Example 8: Mapping of HER3 Epitopes using hydrogen / deuterium exchange mass spectrometry Materials
[0489] [00489] D2O buffer was prepared by dissolving 25 mM TBS (pH 7.5) / 500 mM NaCl in heavy water (Sigma). The reduction solution was 50 mM formate buffer (pH 4) 500 mM TCEP and the 0.5% (v / v) quenching solution of trifluoroacetic acid (TFA) in water. Buffer A was 0.25% formic acid / 10% methanol / 10% ethylene glycol in water, and buffer B was 0.25% formic acid in acetonitrile. All chemicals were purchased from Sigma, and the solvents were HPLC grade from Fisher Scientific. Handling and Liquid Chromatography
[0490] [00490] The experiments of self-hydrogenated hydrogen / deuterium mass spectrometry (MS HDX) were designed based on methods and equipment described by Gales et al., (2006) Anal. Chem. 78: 1005 to 1014). In short, all liquid handling operations used a Pal HTS- (LEAP Technologies) liquid manipulator housed in a refrigerated cabinet maintained at 2 ° C. The injection port valve 6 and a washing station were mounted on the liquid rail and sample injection manipulator facilitated in the chromatographic and syringe washing system. The chromatographic system consisted of an additional 10-port valve, a 2.1 mm x 30 mm Poroszyme pepsin column (Applied Biosystems), a 0.5 mm x 2 mm reverse-phase Cap Trap (Michrom Bioresources) cartridge, and a self-packed electrospray emitter as an analytical column (100 mm x 60 mm ~, Kinetex 2.6 µM C18, Phenomenex). The gate valve head 10, the trap cartridge and the analytical column were housed in a separate box constructed of aluminum and maintained at -5 ° C by Peltier batteries. The valves and columns were configured in such a way as to allow the entry of a digestion chromatography line of the protein, desalination peptide and reverse phase before the introduction of the sample into the ionization source through the electrospray (ESI) of the mass spectrometer (LTQ-Orbitrap, Thermo Scientific).
[0491] [00491] The fluid streams needed for the operation were provided by two pumps separated by HPLC. The first HPLC (Surveyor MS pump, Thermo Scientific) delivered buffer A, at a constant flow of 125 ul / min and was used to transfer the sample through the pepsin cartridge immobilized on the reverse phase trap cartridge, mounted on the other side of the 10 port valve. After the loading and desalination period, port valve 10 was transferred to elute the sample with the help of a gradient pump (AQUITY UPLC, Waters) from the reverse phase trap cartridge, through the analytical column and in the ion source of the mass spectrometer. The immobilized enzyme cartridge was isolated to waste during the gradient elution. The gradient pump delivered the linear gradient segments from 0 to 40% of mobile phase B over 35 minutes at 5 µL / min and 40 to 95% mobile phase B in 5 mL / min over 10 minutes. The flow from the gradient pump was divided into port valve 10 using a passive divider so that the actual flow through the trap cartridge and analytical column for gradient elution was ~ 1 ml / min. The entire chromatographic run was 70 minutes long, including washing and equilibration steps. Mass spectrometry
[0492] [00492] For the purpose of identifying the proteolytic fragments resulting from digestion in various strains of MS / MS dependent data, experiments were carried out. For these in the present acquisitions, the MS tandem spectra were acquired with the LTQ analyzer of the LTQ Orbitrap-hybrid mass spectrometer. The selection of precursor mass was based on DM exams acquired by the Orbitrap analyzer. The acquisitions made in a single phase of MS with the objective of determining the level of deuteration were acquired in a resolution of 60,000 by the Orbitrap analyzer (more than m / z 400 to 2000). Protein and protein preparation: Fab complexes
[0493] [00493] The HER3 protein was prepared by diluting 50 μg HER3-Tag with 25 mM TBS (pH 7.5) / 500 mM NaCl, to obtain a final volume of 50 μl. Protein: The Fab complexes were prepared by mixing 50 µg of the HER3-Tag in a 1: 1 molar ratio with the Fab are studied. Protein: the Fab mixtures were then diluted to a final volume of 50 ml with 25 mM TBS (pH 7.5) / 500 mM NaCl.
[0494] [00494] Protein: Fab complexes were prepared and left to incubate for at least 2 hours at 4 ° C. Four 96-well plates containing samples, quenching solutions, thinner, and reduction were loaded into the liquid handler, before the start of each experiment. For the exchange of experiments 50 µl of HER3 or HER3: Fab complex was diluted with 150 µl of D2O buffer. The mixture was reduced by adding 200 µL of reduction buffer for 1 minute before quenching with 600 µL of quenching buffer. The total volume after all liquid handling steps was ~ 1 ml. Once mixed, the neutralized solution was injected into the chromatographic system where it was automatically digested, separated and analyzed by LCMS. The average variation in deuteration between the sample and the control was calculated as the difference between the levels of deuterium uptake in the sample and the control. Data processing
[0495] [00495] RAW Orbitrap files were converted to mzXML files using a program at home (RawXtract). Subsequently, MS tandem acquisitions were searched using Sequest (Yates Lab, Scripps Research Institute, La Jolla, CA) and the search results were automatically filtered using DTASelect 2.0 (Yates Lab, Scripps Research Institute, La Jolla, CA). Using peptide sequence identifications, an internal written program was used to automatically extract a single ion from chromatograms for each identified sequence and generate average spectra across the entire chromatographic peak. The medium spectra were smoothed and centroidified. The level of deuterium uptake was taken as the difference in mass between a deuterated and non-deuterated reference sample. The manually processed data has been validated and adjusted to correct inaccuracies and errors in automated processing steps. The levels of deuterium uptake were attributed to each of the residues in the protein sequence by relocating the deuterium content in each peptide (that is, it divides the observed deuteration level by the number of amino acids in which the peptide). If a residue was covered by more than one peptide, the normalized deuterium absorptions of all peptides covering the residue were calculated. Example 9: Determination of the X-ray crystallographic structure of the human HER3 / MOR09823 Fab and human HER3 / MOR09825 Fab complexes
[0496] [00496] The present example shows the full length HER3 crystal structure linked with the Fab fragment of MOR09823 and the Fab fragment of MOR09825, determined in the resolution and 3.2a 3.4a, respectively. The labeled human HER3 were further purified on a Hilaad 26/60 Superdex PrepGrade 200 column (GE Healthcare), balanced in PBS (pH 7.3). E. coli and MOR09823 MOR09825 expressed Fabs were isolated by lysing the cells with lysozyme and Fab His6-tag fragments were captured on a HisTrap_HP column (GE Healthcare). The MOR09823 Fab fragments were further purified by means of gel filtration chromatography using a Superdex 75 16/60 column (GE Healthcare), equilibrated in 25 mM Tris (pH 7.5), 150 mM NaCl.
[0497] [00497] The HER3 Fab complexes were prepared by mixing with excess HER3 Fab labeled in a molar ratio of 1.3 to 1.8: 1 (concentration estimated through absorbance at 280 nm, using calculated extinction coefficients of 0.9 and 1.4 (mg / ml) -1cm-1 for HER3 and Fab, respectively) and the purification of the complexes of a 200 10/300 Superdex column (GE Healthcare), balanced in 25 mM Tris (pH 7.5), NaCl at 150 mM. The peak fractions were analyzed by SDS-PAGE and LCMS. For each complex, fractions containing both HER3 and Fab in an approximate equimolar proportion were pooled and concentrated. The HER3 / MOR09823 crystals were grown at 293 K by diffusing a drop of steam from drops containing 150 nl of HER3 / MOR09823 complex and 150 nl of reservoir solution (100 mM sodium citrate pH 5.6, 20 % PEG 4000 and 20% isopropanol). The crystals were transferred to the reservoir solution containing 8% glycerol and cooled instantly in liquid nitrogen. The HER3 / MOR09825 crystals were cultured at 293 K by means of sitting diffusion of steam drop from drops containing 150 nl HER3 / MOR09825 complex and 150 nl of reservoir solution (100 mM bis-tris pH 6.5, 16 % PEG 10000). The crystals were transferred to bis-tris at 100 mM pH 6.5, 18% PEG 10,000 and 22% glycerol and cooled instantly in liquid nitrogen.
[0498] [00498] Data were collected on the light line 17-ID at the Advanced Photon Source (Argonne National Laboratory). The data for the HER3 / MOR09823 Fab complexes were processed and scaled in 3.2a using HKL2000 (HKL Research Inc) in the I222 group space with cell dimensions a = 124.16, b = 139.44, c = 180.25 Â, with good statistics. The Fab HER3 / MOR09823 structure was resolved by molecular substitution using Phaser (McCoy et al., (2007) J. Appl. Cryst. 40: 658 to 674) with fragments of a Fab and the published HER3 of the 1 mb6 structure of DPI as research models. The final model, which contains a molecule of the Fab HER3 / MOR09823 complex by means of the asymmetric unit, was built in COOT (Emsley & Cowtan (2004) Acta Cryst. 60: 2126 to 2132) and refined to R and Rfree values of 19 , 0 and 24.5%, respectively, with an RMSD of 0.010 Â and 1.37 ° for the connection lengths and connection angles, respectively, using BUSTER (Global Phasing, LTD). The HER3 residues, which contain the atoms within 5A of any atom in Fab MOR09823, as indicated in PyMOL (Schrödinger, LLC) are listed in Tables 11 and 12. The data for the HER3 / MOR09825 Fab complexes were processed and scaled in 3.4a using autoPROC (Global Phasing, LTD) in group I222 with cell dimensions a = 124.23, b = 140.94, c = 180.25 Â, with good statistics. The Fab HER3 / MOR09825 structure was resolved by means of molecular substitution using Phaser (McCoy et al., (2007) J. Appl. Cryst. 40: 658 to 674) with the Fab HER3 / MOR09823 structure as a search model. The final model, which contains a molecule of the Fab HER3 / MOR09825 complex by means of the asymmetric unit, was built in COOLT (Emsley & Cowtan (2004) Acta Cryst. 60: 2126 to 2132) and refined to R and Rfree values of 18 , 8 and 24.9%, respectively, with an RMSD of 0.009 Â and 1.21 ° for the connection lengths and connection angles, respectively, using BUSTER (Global Phasing, LTD). The HER3 residues, which contain atoms within 5A of any atom in Fab MOR09825, as indicated in PyMOL (Schrödinger, LLC) are listed in Tables 13 and 14. Example 10: Phospho-HER3 in in vitro cell assays.
[0499] [00499] MCF-7 cells were usually maintained in DMEM / F12, 15mM HEPES, L-glutamine, 10% FCS and SK-BR-3 in McCoy's 5a, 10% FCS, 1.5 L-glutamine mM. Sub-confluent MCF7 or SK-BR-3 from cells cultured in complete medium were harvested with Accutase (PAA Laboratories) and resuspended in the appropriate growth medium at the final concentration of 5 x 105 cells / ml. 100 µl of cell suspension was then added to each well of a 96-well flat bottom plate (Nunc), to give a final density of 5 x 104 cells / well. MCF7 cells were allowed to adhere for approximately 3 hours before the medium was exchanged for starvation, containing 0.5% FBS. All plates were then incubated overnight at 37 ° C prior to treatment with the appropriate concentration of HER3 Antibodies (diluted in appropriate media) for 80 minutes at 37 ° C. MCF7 cells were treated with 50 ng / mL 1-β1 neuregulin from the EGF domain (R & D Systems) for the final 20 minutes to stimulate HER3 phosphorylation. All media was carefully aspirated and the cells were washed with ice-cold PBS containing 1 mM CaCl2 and 0.5 mM MgCl2 (Gibco). The cells were lysed by adding 50 μl of ice-cooled lysis buffer (20 mM Tris (pH 8.0) / 137 mM NaCl / 10% glycerol / 2 mM EDTA / 1% NP-sodium a 40 mM / 1 orthovanadate /, aprotinin (10 µg / mL) / Leupeptin (10μg / mL)) and incubated on ice, with shaking, for 30 minutes. The lysates were then collected and centrifuged at 1800 g for 15 minutes at 4 ° C to remove cell debris. 20 µl of lysate was added to a pre-prepared capture plate.
[0500] [00500] The HER3 capture plates were generated using a carbon plate (Mesoscale Discovery) coated overnight at 4 ° C with 20 µL of 4 µg / mL MAB3481 capture antibody (R & D Systems) diluted in PBS and subsequently blocked with 3% bovine serum albumin in 1x Tris buffer (Mesoscale Discovery) / 0.1% Tween-20. HER3 was captured from the lysate by incubating the plate at room temperature for one hour with shaking before the lysate was aspirated and the wells washed with 1x Tris buffer (Mesoscale Discovery) / 0.1% Tween-20. Phosphorylated HER3 was detected using 0.75 µg / mL of biotinylated anti-phosphotyrosine antibody (R & D Systems), prepared in 1% BSA / 1x Tris / 0.1% Tween-20 by incubation with shaking at temperature room for 1 hour. The wells were washed four times with 1x Tris / 0.1% Tween-20 protein and biotinylated were detected by incubation with S-Tag Streptavidin labeled (Mesoscale Discovery) diluted in 1% BSA / 1x Tris / 0.1 % Tween-20 for one hour at room temperature. Each well was aspirated and washed four times with 1x Tris / 0.1% Tween-20 before adding 20 µl of T Ler buffer with surfactant (Mesoscale Discovery) and the signal quantified using a Sector Mesoscale image generator. MOR06391 or MOR03207 antibodies were included in signaling experiments as isotypic controls. Example 11: phospho-Akt (S473) in in vitro cell assays.
[0501] [00501] Sub-confluent of SK-BR-3 and BT-474 cells cultured in complete medium were harvested with Accutase (PAA Laboratories) and resuspended in the appropriate growth medium at the final concentration of 5 x 105 cells / ml. 100 µl of the cell suspension was then added to each well of a 96-well flat bottom plate (Nunc), to obtain a final density of 5 x 104 cells / well. All plates were then incubated overnight at 37 ° C prior to treatment with the appropriate concentration of HER3 Antibodies (diluted in appropriate media) for 80 minutes at 37 ° C. All media was carefully aspirated and the cells were washed with ice-cold PBS containing 1 mM CaCl2 and 0.5 mM MgCl2 (Gibco). The cells were lysed by adding 50 μl of ice-cooled lysis buffer (20 mM Tris (pH 8.0) / 137 mM NaCl / 10% glycerol / 2 mM EDTA / 1% sodium orthovanadate NP-40 mM / 1 / aprotinin (10μg / mL) / Leupeptin (10μg / mL)) and incubated on ice, with shaking, for 30 minutes. The lysates were then collected and centrifuged at 1800 g for 15 minutes at 4 ° C to remove cell debris. 20 µl of lysate was added to a 384 multi-well Phospho-Akt carbon plate (Mesoscale Discovery) site that had previously been blocked with 3% BSA / 1x Tris / 0.1% Tween-20. The plate was incubated at room temperature for two hours with shaking before the lysate was aspirated and the wells washed four times with 1x Tris buffer (Mesoscale Discovery) / 0.1% Tween-20. Phosphorylated Akt was detected using 20 µL of anti-phospho-Akt SULFO-LABEL (S473) antibody (Mesoscale Discovery) diluted 50 times in 1% BSA / 1x Tris / 0.1% Tween-20 by means of incubation with shaking at room temperature for 2 hours. The wells were washed four times with 1x Tris / 0.1% Tween-20 before adding 20 µl of T Ler buffer with surfactant (Mesoscale Discovery) and the signal quantified using a Sector Mesoscale image generator. MOR06391 or MOR03207 antibodies were included in signaling experiments as isotypic controls. Example 12: Proliferation cell line assays.
[0502] [00502] SK-BR-3 cells were routinely cultured in modified McCoy 5A medium, supplemented with 10% fetal bovine serum and 474 BT-cells were cultured in DMEM supplemented with 10% FBS. Sub-confluent cells were trypsinized, washed with PBS, diluted to 5x104 cells / ml with growth medium and plated in 96-well plates with black transparent background (Costar 3904), at a density of 5000 cells / well. The cells were incubated overnight at 37 ° C before adding an appropriate concentration of HER3 antibody (typically final concentrations of 10 or 1 µg / ml). The plates were returned to the incubator for 6 days before assessing cell viability using CellTiter-Glo (Promega). 100 L of CellTiter-Glo solution was added to each well and incubated at room temperature with gentle shaking for 10 minutes. The amount of luminescence was determined using a SpectraMax plate reader (Molecular Devices). The extent of growth inhibition obtained with each of the antibodies was calculated by comparing the values obtained in each HER3 luminscence antibody to a standard isotype control antibody (MOR06391).
[0503] [00503] For the proliferation assays of MCF-7 cells were routinely cultured in DMEM / F12 (1: 1) containing 4 mM L-glutamine / 15 mM at 10% HEPES / FBS. Sub-confluent cells were trypsinized, washed with PBS and diluted to x105 cells / ml with DMEM / F12 (1: 1) containing 4 mM L-glutamine / 15 mM HEPES / 10 µg / ml human transferrin / BSA a 0.2%. The cells were plated in 96-well plates with a transparent black background (Costar) at a density of 5000 cells / well. The appropriate concentration of HER3 antibody (typical final concentrations of 10 or 1 µg / ml) was then added. 10 ng / ml of EGF NRG1-β1 domain (R & D Systems) was also added to the appropriate wells to stimulate cell growth. The plates were returned to the incubator for 6 days before assessing cell viability using CellTiter-Glo (Promega). The extent of growth inhibition obtained with each of the antibodies was calculated by subtracting the background (non-neuregulin) luminscence values and comparing the resulting values obtained with each of the anti-HER3 antibodies to a standard isotype control antibody ( MOR06391). Example 13: Cellular ligand blocking assays
[0504] [00504] MCF-7 cells cultured in MEM supplemented with 10% FBS and 1 µg of insulin / mL (Sigma) were washed and collected in a small volume of FACSmax cell dissociation buffer (Genlantis) before adding 5 mL of FACS buffer (PBS / FBS 1% sodium azide / 0.1%). The cell density was counted and adjusted to a final concentration of 1 x 106 cells / ml. 100 µl of cell suspension was added to each well of a 96 well plate and the cells pelleted by centrifugation (220 g, 3 minutes, 4 ° C). Cell pellets were resuspended in 100 µl of suitable assay antibodies diluted in FACS buffer (typical final concentrations of antibodies ranged from 100 to 0.1 nM) and the plate was incubated on ice for 45 minutes. The MAB3481 blocking antibody ligand (R & D Systems) was included as a positive control. The cells were washed twice with staining buffer before adding 10 nM NRG1-β1 EGF domain (R & D Systems) diluted in FACS buffer and incubating on ice for 45 minutes. The cells were washed twice with stain buffer and bound neuregulin detected by incubating the cells with 10 nM anti-human NRG1-β1 EGF domain antibodies (R & D Systems) on ice for 45 minutes. The cells were washed twice with staining buffer and incubated on ice for 45 minutes with PE-bound anti-goat antibody (Jackson ImmunoResearch) diluted 1/500 with FACS buffer. The cells were then pelleted by centrifugation and the pellet resuspended in 200 µl of FACS buffer. To quantify each sample 10,000 live cells were counted on an LSR Flow Cytometer II (BD Biosciences) and the amount of cell surface bound to neuregulin was assessed by measuring the mean channel fluorescence. Example 14: Biochemical assays for ligand blocking
[0505] [00505] The method of the present invention includes a biosensor-based plasmid surface resonance (SPR) utility (BiacoreTM, GE Healthcare, Uppsala, Sweden), to examine the ability of HER3 / antibody to bind to neuregulin complexes.
[0506] [00506] BiacoreTM uses the plasmonic surface resonance (SPR) phenomenon to detect and measure the binding interaction. In a typical Biacore experiment, one of the interacting molecules (neuregulin) is immobilized in a matrix, while the interaction partner (HER3) flows over the surface. The interaction link results in an increase in mass on the sensor surface and the corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in the index of refraction or signal are recorded in resonance units (UK) due to changes in the association and dissociation signals of complexes are monitored non-invasively, continuously and in real time, the results of which are presented under the shape of a sensorgram.
[0507] [00507] BiacoreTM T100 (GE Healthcare, Uppsala, Sweden) was used to carry out all the experiments reported in the present invention. Preparation of the surface sensor and analyzes were performed using the 250C interaction. Buffer and Biacore reagents were purchased from GE Healthcare. Running buffer containing 10 mM Hepes, pH 7.4 / 150 mM NaCl, 0.05% P20, 0.5% BSA was used throughout the trial.
[0508] [00508] The extracellular domain NRG-1β1 (R & D Systems) was incubated on ice for 45 minutes with EZ-link Sulfo-NHS-LC-LC-Biotin (Pierce) at a molar ratio of 5: 1. The reaction was quenched by adding excess ethanolamine and uncoupled biotin removed from biotinylated-NRG using centrifugation columns (desalting Zeba). Biotinylated-NRG was captured on a pre-immobilized CAP sensor chip with approximately 3000 RU of ssDNA-streptavidin (Biotin capture kit) to produce neuregulin surface densities in the range of 400 to 600 RU The flow cell was generated using the reference omitting biotinylated-NRG from the injection steps in such a way that only DNA-streptavidin was present on the surface of the flow cell.
[0509] [00509] HER3 / antibody complexes were generated by incubating 10 nM human HER3-Fc with increasing concentrations (0 to 50 nM) of the appropriate test antibody for 15 minutes at room temperature, before incubation at 10 ° C BiacoreTM ° C. The analyzes were performed by interaction injecting HER3 / reference antibody and the complexes plus neuregulin surfaces in series for 180 seconds at a flow rate of 60 μL / min. The dissociation complex was monitored for 180 seconds at a flow rate of 60 μL / min. Surface regeneration was performed at the end of each analysis cycle using an injection of 120 seconds 8M guanidine: 1 M NaOH (3: 1) followed by a second injection of 120 30% acetonitrile / NaOH 0.25 M, at a flow rate of 30 μL / min. Example 15: In vivo PD studies
[0510] [00510] BxPC3 and BT-474 cells were cultured and implanted in athymic nu / nu Balb / C females (Harlan Laboratories) as described in Examples 16 and 17.
[0511] [00511] Once the tumors had reached an appropriate size, the animals were examined for tumor quality. Animals with ulcerated tumors or animals with fluid-filled tumors were excluded from the study. The remaining animals were dosed intravenously with antibodies by injection into the lateral tail vein. At the determined time points, the animals were sacrificed by means of CO2 asphyxiation and whole blood was collected through cardiac puncture and placed in a 1.5 ml Eppendorf collection tube. The tumor tissue was immediately dissected, placed in a polypropylene screw cap sample tube and frozen in liquid nitrogen. The tissue was stored at -80 ° C until lysates were prepared. Example 16: BT-474 in vivo efficacy studies
[0512] [00512] BT-474 cells were cultured in DMEM containing 10% serum inactivated by fetal bovine heat without antibiotics, until the moment of implantation.
[0513] [00513] The day before the cells were inoculated, the athymic nu / nu Balb / C females (Harlan Laboratories) were implanted subcutaneously with a sustained release of 17 β-estradiol pellets (Innovative Research of America), to maintain levels of estrogen in the serum. One day after implantation of the β 17-estradiol pellet, 5 x106 cells were injected orthotopically into the mammary fatpad 4 in a suspension containing 50% phenol red free matrigel (BD Biosciences) in Hank's balanced saline solution. The total injection volume, containing the cells in suspension, was 200 μL. 20 days after implantation of animal cells, with a tumor volume of approximately 200 mm3, they were enrolled in the efficacy study. In general, a total of 10 animals per group were included in efficacy studies.
[0514] [00514] For the single agent studies, the animals were dosed intravenously through injection into the lateral tail vein, with either MOR10701 or MOR10703. An initial loading dose of 40 mg / kg was administered during the first dose. After the initial dose, the animals were on a 20 mg / kg schedule every two days for the duration of the study. For combination studies, animals were dosed with either MOR10701 or MOR10703 (20mg / kg, iv, q2d) and a sub-optimal dose of trastuzumab (1mg / kg, iv, 2QW).
[0515] [00515] For the duration of the studies, the tumor volume was measured by calipering twice a week. Treatment / control percentages (T / C) were calculated using the following formula: % T / C = 100 '... T / ... C if ... T> 0
[0516] [00516] where:
[0517] [00517] T = mean tumor volume of the drug-treated group on the final day of the study;
[0518] [00518] ... T = mean tumor volume of the drug treated group on the final day of the study - mean tumor volume of the drug treated group on the initial dosing day;
[0519] [00519] C = mean tumor volume of the control group on the last day of the study, and
[0520] [00520] ... C = mean tumor volume of the control group on the last day of the study - mean tumor volume of the control group on the initial dosing day.
[0521] [00521] Body weight was measured twice a week and dose was adjusted body weight. The% change in body weight was calculated as (BWcurrent - BWinitial) / (BWinitial) x 100. The data are presented as a percentage of change in body weight on the day the treatment starts.
[0522] [00522] All data were expressed as mean ± standard error of the mean (SEM). Delta tumor volume and body weight were used for statistical analysis. Between groups of comparisons were performed using a one-way ANOVA followed by a Tukey post hoc. For all statistical assessments, the level of significance was set at p <0.05. Meaning compared to the vehicle control group is reported. Example 17: BxPC3 in vivo efficacy studies
[0523] [00523] BxPC3 cells were cultured in RPMI-1640 medium containing 10% serum inactivated by fetal bovine heat without antibiotics, until the moment of implantation.
[0524] [00524] The female athymic nu / nu Balb / C females (Harlan Laboratories) were implanted subcutaneously with 10 x106 cells in a mixture of 50% phosphate buffered with 50% matrigel. The total injection volume, containing the cells in suspension, was 200 μL. Once the tumors had reached approximately 200mm3 in size, the animals were enrolled in the efficacy study. In general, a total of 10 animals per group were included in the studies. Animals were excluded from enrollment if they exhibited unusual tumor growth characteristics prior to enrollment.
[0525] [00525] The animals were dosed intravenously through the lateral injection tail vein. An initial loading dose of 40 mg / kg was administered during the first dose. After the initial dose, the animals were at a 20 mg / kg, alternating schedule days for the duration of the study (25 days of treatment). The tumor volume and T / C values were calculated as previously detailed. Example 18: In vivo DP assays with phospho-Akt (S473).
[0526] [00526] Approximately 50 mm3 of the frozen tumor (for example, BT-474 or BXPC-3) tissue was thawed on ice and 100 - 300 µl of T-PER buffer (Pierce) containing phosphatase (Roche) and protease inhibitors (Roche) was added to each sample. The volume of lysis buffer added was dependent on the size of the tumor sample. The tissue was divided using a 1.5 ml pestle (Fisher Scientific) and the resulting suspensions were incubated on ice for 15 minutes before being frozen overnight at -80 ° C. The samples were thawed and centrifuged for 15 minutes at 13000 g, 4 ° C, before the quantification of the supernatant protein content by BCA assay (Thermo Scientific). Tissue supernatants were diluted with lysis buffer (Mesoscale Discovery) and 25 µg added to a 96-well Phospho-Akt site on the carbon plate (Mesoscale Discovery) that had previously been blocked with A-blocking solution. (Mesoscale Discovery) The plate was incubated at room temperature for one hour with shaking before the lysate was aspirated and the wells were washed four times with Tris wash buffer (Mesoscale Discovery). Phosphorylated Akt was detected using 25 µl of anti-phospho-Akt SULFO-LABEL (S473) antibody (Mesoscale Discovery) diluted in antibody dilution buffer, by incubating with shaking at room temperature for one hour. The wells were washed four times with Tris wash buffer before adding 150 µL of reading T buffer (with surfactant) (Mesoscale Discovery) and the signal quantified using a Sector Mesoscale image generator. Example 19: PD in vivo assays with Phospho HER3 (Y1197)
[0527] [00527] Approximately 50 mm3 frozen tumor (eg, BXPC-3) tissue was thawed on ice and 100 - 300 µl of T-PER buffer (Pierce) containing phosphatase (Roche) and protease inhibitors (Roche) was added each sample. The tissue was divided using a 1.5 ml pestle (Fisher Scientific) and the resulting suspensions were incubated on ice for 15 minutes before being frozen overnight at -80 ° C. The samples were thawed and centrifuged for 15 minutes at 13000 g, 4 ° C, before the quantification of the supernatant protein content by BCA assay (Thermo Scientific). Tissue supernatants were diluted with lysis buffer and 150 µg added to a 96-well multi-plate carbon site (Mesoscale Discovery) that had previously been coated overnight with 4 µg / ml MAB3481 (R & D Systems) and blocked with 3% milk. The plate was incubated at room temperature for two hours with shaking before the lysate was aspirated and the wells were washed four times with Tris wash buffer (Mesoscale Discovery). Phosphorylated HER3 was ligated using anti-HER3 pY1197 diluted 1/8000 with blocking buffer. After incubation at room temperature for one hour, the wells were washed with Tris wash buffer and anti-pY1197 antibody detected using anti-rabbit labeled S-Tag antibody (Mesoscale Discovery) diluted 1/1000 in blocking buffer, by incubating with shaking at room temperature for one hour. The wells were washed four times with Tris wash buffer before adding 150 µl of 1/4 diluted Read T buffer (with surfactant) (Mesoscale Discovery) and the signal quantified using a Sector Mesoscale image generator. Example 20: In vitro drug combination studies
[0528] [00528] To assess the ability of target HER3 antibodies to combine with targeted therapies MOR09825 or MOR10703 were combined with trastuzumab, lapatinib, BEZ235, BKM120, BYL719, RAD001, erlotinib and cetuximab in cell viability assays. Approximately 1000 to 1500 SK-Br-3 (McCoy), MDA-MB-453 (RPMI), FADU (EMEM) or L3.3 (RPMI), cells were seeded in 384 well plates in suitable culture media supplemented with 2% FBS and allowed to adhere overnight at 37 ° C. Suitable drug combinations (typical final drug concentrations for lapatinib, BKM120 and BYL719 ranged from 3μΜ to 13 nM; for RAD001 it ranged from 27NM to 0.0041nM; for erlotinib it ranged from 1 µM to 0.0025nM; for MOR1073 it ranged from 100 nm to 0.01nm; for cetuximab it varied from 100 nM to 0.0015nM, and for trastuzumab it was 300 nM to 0.046nM)) were later added to the wells so that each plate contained a dose-response curve for each drug in a matrix two-dimensional. The plates were returned to the incubator for 3 to 6 days before assessing cell viability using CellTiter-Glo (Promega). CellTiter-Glo solution was added to each well and incubated at room temperature with gentle shaking for 10 minutes. The amount of luminescence was determined using a SpectraMax plate reader (Molecular Devices). The extent of growth inhibition obtained with each combination was calculated and combination activity was enhanced using the Loewe additivity model. Example 21: In vivo drug combination studies in L3.3 cells
[0529] [00529] Pancreatic L3.3 cells were cultured in DMEM medium containing 10% inactivated by the heat of fetal bovine serum, until the moment of implantation. Foxn1 nude female mice (Harlan Laboratories) were implanted subcutaneously with 3 x 106 cells in FBS-free DMEM. The total injection volume, containing the cells in suspension, was 100 μL. 12 days after cell implantation, the animals were enrolled in the efficacy study, with an average tumor volume of approximately 100mm3 for all groups. In general, a total of 8 animals per group were included in the studies. Animals were excluded from enrollment if they exhibited unusual tumor growth characteristics prior to enrollment.
[0530] [00530] The animals were dosed intravenously with MOR10703 through injection into the lateral tail vein, at 20 mg / kg, on alternate days schedule for the duration of the study (14 days of treatment). Erlotinib was administered in doses of 50mg / kg (PO) on a daily schedule, either as a single agent or in combination with MOR10703. The tumor volume and T / C values were calculated as previously detailed. Results and discussion
[0531] [00531] Collectively, these results show that a class of antibodies bind to amino acid residues within domain 2 and domain 4 of a HER3 conformational epitope and stabilize HER3 in an inactive or closed conformation. The binding of these antibodies inhibits both ligand-dependent and ligand-independent signaling. These antibodies are also capable of binding simultaneously with an HER3 linker. Affinity Determination
[0532] [00532] Antibody affinity was determined by titrating the equilibrium solution (SET), as described above. The results are summarized in Table 9 and example titration curves for MOR10701 are contained in Figure 1. The data indicates that a number of antibodies have been identified that are closely bound to human, cino, rat and mouse HER3. Table 9: KD values of anti-HER3 IgGs as determined by titrating an equilibrium solution (SET). Hu (human), Ci (cinomolgo), Mu (murine) and ra (rat)
[0533] [00533] The ability of the identified antibodies to bind cells expressing HER3 was determined by calculating the EC50 values for their binding to the SK-Br-3 amplified HER2 cell line (see Figure 2 and Table 10). Table 10: EC50 values of anti-HER3 IgG FACS in SK-BR-3 cells. na (not determined)
[0534] [00534] A subset of anti-HER3 antibodies was characterized by their ability to bind the various extracellular domains of human HER3 in an ELISA assay. To achieve this goal, the extracellular domain of HER3 has been divided into its four constituent domains and various combinations of these domains have been cloned, expressed and purified as independent proteins, as described above. Using this strategy, the following domains have been successfully generated as soluble proteins: domains 1 and 2 (D1-2), domains 2 (D2), domains 3 and 4 (D3-4) and domain 4 (D4 ). A number of HER3 Antibodies generated internally from anti-human mice (8D7, 1F5 and 8P2) were also tested as positive controls to demonstrate the integrity of each isolated domain.
[0535] [00535] As shown in Figure 3 and MOR09823 MOR09825 were observed to both successfully bind the HER3 extracellular domain, but little binding to isolated domains was observed in this assay with these antibodies. There are several possible explanations for this link pattern:
[0536] [00536] a) MOR09823 and MOR09825 can bind a linear epitope that spans a domain boundary, therefore, part of the binding epitope would be lost when the domains were expressed as isolated proteins.
[0537] [00537] b) MOR09823 and MOR09825 can link a non-linear epitope that links several domains. Consequently, the separation of HER3 into its component units can destroy the binding site.
[0538] [00538] c) The shape / conformation of HER3 can be a component of the connection of MOR09823 and MOR09825 to HER3 in such a way that only the extracellular full-length domain of HER3 is able to adopt this shape / conformation, while the isolated domains they cannot fully assume this conformation. (Vi) Mapping of HER3 Epitopes in the use of hydrogen / deuterium exchange mass spectrometry
[0539] [00539] The HER3 epitope was explored through the HDX-MS analysis of HER3 ECD, in the presence and absence of Fab versions MOR09823, MOR09824, MOR09825 and MOR09974. Figure 4A shows that, in the absence of bound Fab, approximately 69% of the HER3 ECD sequence was covered by at least one peptide. Coverage gaps may be due to glycosylation of residues within these regions or insufficient reduction of cysteine disulfide bonds in rich regions, which is particularly evident in domain 2. Interestingly, although each Fab produced individual protection standards, a region of strong protection has been observed consistently with MOR09823, MOR09824, MOR09825 and MOR09974 (see Figure 4B), indicating that these highly related family of antibodies bind HER3 in an identical manner. The strongest was observed for the protection of domain 2 residues 269-286 (TFQLEPNPHTKYQYGGVC) (SEQ ID NO: 146), indicating that residues in this vicinity may be important for mAb binding. Mapping of protected Fab residues to the published crystal structure HER3 (Cho & Leahy, (2002) Science 297: 1330 to 1333) points out that the 269 to 286 residues are within and proximal to a functionally important β-hairpin loop within domain 2 ( see Figure 4C). (Vii) crystal structure HER3 / MOR09823
[0540] [00540] Resolution 3.2A MOR09823 x-ray crystal structure of the Fab fragment linked to the HER3 extracellular domain has been resolved to better define the HER3 epitope, which is recognized by this family of related antibodies (see Figure 5A). In addition, the structure of MOR09825 3.4a of the Fab fragment bound to human HER3 has been resolved. In both MOR09823 / HER3 and MOR09825 / HER3 crystalline structures, HER3 is in the tethered (inactive) conformation (see Figure 5A, B, C and D). This conformation is characterized by a significant interaction interface between domains 2 and 4, mediated by a β-hairpin dimerization circuit in domain 2. The observed conformation of HER3 is similar to that previously described by Cho et al. (Cho & Leahy, (2002), Science 297: 1330 to 1333), who published the crystalline structure of the HER3 extracellular domain in the absence of neuregulin. Since neuregulin can activate HER3, the conformation of tethered HER3 is presumed to be inactive. Similar captive conformations were also observed when related family members EGFR HER4 (Bouyain et al., (2005) Proc. Natl. Acad. Sci. US, 102: 15024 to 15029) and HER1 (Ferguson et al., (2003 ) Molec. Cell 11: 507 to 517) were crystallized.
[0541] [00541] The spatial relationships between domains 1 to 4 of HER3 in the inactive (tethered) state are significantly different from that of the extended (active) state. This conclusion is based on the crystalline structures of related members of the EGFR family and HER2-linked ligand HER1 (Cho et al, (2003) Nature 421: 756 to 760;. Ogiso et al, (2002) Cell 110: 775 to 787 , Garrett et al, (2002) Cell 110: 763 to773) both of which are in an extended (active) state. In the extended state, the dimerization domain of the β-hairpin loop 2 is freed from its inhibitory interaction with 4 and is therefore free to interact with its partners dimerization proteins. Thus, the β-hairpin cycle 2 dimerization domain is functionally important both for maintaining the (inactive) tethered state and in mediating the dimerization of EGF receptors in their extended state, leading to the activation of the intracellular kinase domain. The crystalline structures MOR09823 / HER3 and MOR09825 / HER3 (see Figure 5), therefore, suggest that both MOR09823 and MOR09825 function, stabilizing the inactive conformation of HER3.
[0542] [00542] The crystalline structure also revealed that the epitope recognized by both HER3 and MOR09823 MOR09825 is a non-linear epitope that includes residues from both domains 2 and 4 (see Figure 5C and D, Tables 11, 12, 13 and 14 ). The epitope recognized by HER3 this family of highly related antibodies can therefore be defined as:
[0543] [00543] Domain 2: 265 to 277, 315 waste
[0544] [00544] Domain 4: 571,582 to 584, 596 to 597, 600 to 602, 609 to 615 residues
[0545] [00545] The connection of both two domains and 4 by MOR09823 or MOR09825, consequently, to stabilize the tethered conformation of HER3 thus antoganize its ability to signal.
[0546] [00546] The connection mode MOR09823 / MOR09825 observed in the crystalline structure is consistent with our other epitope mapping studies. Specifically, the ELISA experiments of the binding domain show that the affinity of MOR09823 and MOR09825 are significantly higher for the intact extracellular protein HER3 than for any isolated domains (for example, D1, D1-D2, D3, D4 or D3-fragments ) (see Figure 3). There is also an agreement with the HDX-MS HER3 data (see Figure 4B), which identifies the β-hairpin domain 2 as part of the recognition of the antibody epitope. Finally, both crystalline structures indicate that the HER3 ligand-binding surface, which was mapped by analogy with HER1 for domains 1 and 3 (Ogiso et al, (2002) Cell, 110: 775 to 787, Garrett et al, (2002) Cell, 110: 763 to 773), is not obstructed by any MOR09823 or MOR09825 connection (see Figure 5B). This is consistent with our findings that neither MOR09823 nor MOR09825 neuregulin in the MCF7 cell binding block (see Figure 9), and that HER3 / MOR09823 complexes can bind to immobilized neuregulin in BIAcore studies (see Figure 10). Table 11: Interactions between MOR09823 Fab heavy chain and human HER3. Fab VH residues are numbered based on their linear amino acid sequence (SEQ ID NO: 15). HER3 residues are numbered based on NP_001973. HER3 residues shown have at least one atom within 5a of an atom in the MOR09823 Fab.
[0547] [00547] Visual inspection of the MOR09823 / MOR09825 crystal structures highlighted the HER3 Lys267 Leu268 residues formed from multiple interactions with the various CDR antibodies, suggesting that they may be important for antibody binding. Consequently, Lys267 and / or Leu268 were mutated to alanine, expressed and the resulting purified recombinant proteins, in order to assess their impact on antibody binding. Binding ELISA assays indicated that the mutation of Lys267 or Leu268 or abolished MOR10703 binding to HER3 (Figure 5F), suggesting that both residues are an integral part of the HER3 epitope and thus supporting the interactions between proposals MOR09823 / MOR09825 and HER3. (viii) Inhibition of cell signaling
[0548] [00548] To determine the effect of anti-HER3 antibodies on MCF7 HER3 ligand-dependent activities, cells were incubated with IgG prior to neuregulin stimulation. The example inhibition curves are illustrated in Figure 6A and summarized in Table 15. The effect of HER3 anti-HER2 antibodies upon HER3-mediated activation was also studied using the SK-Br-3 amplified HER2 cell line (Figure 6B and Table 15). Table 15: pHER3 IC50 and extent of inhibition values of HER3 anti-IgG antibodies in MCF7 and SK-BR-3 cells.
[0549] [00549] To determine whether inhibition of HER3 activity impacted downstream of the Akt signaling cell, phosphorylation was determined in amplified HER2 cells after treatment with HER3 antibodies (see Figure 7 and Table 16). Table 16: IC50 pAkt (S473) and extent of inhibition values of anti-HER3 IgG antibodies in 3-SK-Br BT-474 and MCF7 cells.
[0550] [00550] In summary MOR09823, MOR09824, MOR09825, MOR09974, MOR10701, MOR10702 MOR10703, MOR12609 and MOR12610 are each capable of inhibiting cellular HER3 activity both in the form of a ligand dependent and ligand independent. (ix) Inhibition of proliferation
[0551] [00551] Since MOR09823, MOR09824, MOR09825, MOR09974, MOR10701, MOR10702 and MOR10703 all inhibited HER3 and downstream signaling activity they have been tested for their ability to block ligand dependent and independent in vitro cell growth (data from example is shown in figure 8 and summarized in Table 17). The anti-HER3 antibodies tested were all effective inhibitors of cell proliferation. Table 17: Inhibition of proliferation after treatment with 10 µg / ml of anti-HER3 IgG in 3-SK-Br, BT-474 and MCF7 cells.
[0552] [00552] The ability of the described anti-HER3 antibodies to block ligand binding has been determined by examining the neuregulin binding to MCF7 cells previously treated with either MOR09823 or MOR09825. The presence of either MOR09823 or MOR09825 had no significant effect on the ability to bind to MCF7 neuregulin cells, while the positive control used in the experiment (Mab3481) was able to interfere with neuregulin binding profoundly (see Figure 9) . These results are consistent with the crystal structure since MOR09823 interacts with domains 2 and 4 whereas the main contact points of interaction with HER3 neuregulin are hypothetically to be mainly grouped within domains 1 and 3. Since neuregulin is capable of if it links the inactive HER3 conformation (Kani et al, (2005) Biochemistry 44: 15842 to 15857), it is likely that the MOR09823 and MOR09825 function prevented the HER3 domain rearrangements necessary for signaling or by interfering with the dimerization receptor. (Xi) Evaluation of blocking ligand (biochemical)
[0553] [00553] To explore whether MOR09823 and neuregulin can simultaneously bind HER3 a biochemical assay was created using BiacoreTM technology. The analyzes were performed through the interaction of the capture of biotinylated neuregulin on the surface of a CAP BiacoreTM sensor chip (GE Healthcare) using a biotin capture kit (GE Healthcare). HER3 complexes were generated by incubating human HER3-Fc with increasing concentrations of either MOR09823, 105.5 (Thermo Scientific) or human IgG. Preformed HER3 / antibody complexes were injected over reference and active surfaces and the interaction of HER3 with observed neuregulin.
[0554] [00554] Control IgG had no effect on HER3 / neuregulin from complex formation, while 105.5 was observed to significantly inhibit the ability to bind HER3 to neuregulin confirming its description as a blocking ligand antibody (Figure 10). In contrast, the HER3 / MOR09823 complexes were able to demonstrate that the neuregulin binding MOR09823 does not prevent ligand binding. Interestingly, a dose-dependent increase in RU values was observed exclusively when the MOR09823 / HER3 complexes were injected. These data indicate that a trimeric complex containing neuregulin, HER3 and MOR09823 is generated on the chip surface. The ability of this trimeric complex to form is predicted by the crystal structure HER3 / MOR09823 since MOR09823 binding does not occlude the binding site of the HER3 ligand, suggesting that the binding of neuregulin and MOR09823 are not mutually exclusive.
[0555] [00555] In another embodiment, the antibody or a fragment of it binds to both domain 2 and domain 4 of HER3 and without blocking the simultaneous binding of a ligand such as the HER3 neuregulin. Although it is not necessary to provide a theory, it is possible that the antibody or a fragment of it binds to domain 2 and domain 4 of HER3, HER3 holds in an inactive conformation, without blocking the ligand binding site in HER3. In this way, an HER3 ligand (for example, neuregulin) is able to bind to HER3, at the same time as the antibody.
[0556] [00556] The antibodies of the present invention, or fragments thereof, inhibit both activation of the ligand-dependent and HER3-independent, without preventing ligand binding. This is considered advantageous for the following reasons:
[0557] [00557] (I) The therapeutic antibody will have clinical utility for a broader spectrum of tumors than an antibody that targets a single HER3 activation mechanism (i.e., ligand independent or ligand-dependent), since different types of tumors are triggered through each mechanism.
[0558] [00558] (Ii) The therapeutic antibody would be effective in tumor types in which both HER3 activation mechanisms are simultaneously involved. An antibody targeting a single HER3 activation mechanism (i.e., ligand-dependent or ligand-independent) that exhibits little or no efficacy in these types of tumors
[0559] [00559] (Iii) The effectiveness of an antibody that inhibits ligand-dependent activation of HER3 without preventing ligand binding would be less likely to be adversely affected by increasing ligand concentrations. This implies either greater efficiency in a type of tumor driven by very high concentrations of HER3 ligand or reduced drug resistance, where resistance is mediated by over-regulation of HER3 ligands.
[0560] [00560] (Iv) an antibody that inhibits activation by HER3 stabilizes the inactive form that would be less prone to resistance to drugs triggered through the alternative mechanisms of HER3 activation.
[0561] Consequently, the antibodies of the present invention can be used to treat conditions in which existing therapeutic antibodies are clinically ineffective. (Xii) In vivo inhibition of HER3 activity and effect on tumor growth
[0562] [00562] To determine the in vivo activity of the described anti-HER3 antibodies, MOR09823 was tested on both BxPC-3 and BT-474 tumor models. MOR09823, has been shown to inhibit HER3 activity as evidenced by a significant reduction in pHER3 tumor levels (Figure 11). Downstream of HER3 signaling was similarly inhibited, as demonstrated by the reduced levels of both pAkt BxPC-3 and BT-474 (Figure 11). In a HER2-oriented BT-474 efficacy study, repeated treatment MOR10701 produced 74% inhibition of tumor growth (see Figure 12A), while MOR10703 produced 83% inhibition. In the BxPC3 growth tumor model, both MOR10703 and MOR10701 very effectively inhibited the growth of the targeted ligand tumor (see Figure 13). (Xiii) The combination of drugs in vitro and impact on cell growth.
[0563] [00563] Since the growth of tumor cells is often driven by multiple signaling pathways it was assessed whether combinations of MOR09823 or MOR10703 with various target agents would be of benefit in blocking cell proliferation. The targeted agents chosen mainly inhibited HER2 (trastuzumab, lapatinib) EGFR (cetuximab, erlotinib), PI3K / mTOR (BEZ235), PI3K (BKM120), PIK3CA (BYL719) and mTOR (RAD001) since these targets are normally activated in tumors humans. Isobologram analysis (see Figure 14) indicated that MOR09823 and MOR10703 exhibited synergistic drug combinations with trastuzumab, lapatinib, erlotinib, cetuximab, BEZ235, BZM120, BYL719 and RAD001. These data suggest that inhibition of HER3 signaling is particularly advantageous for inhibitors targeting tyrosine kinase receptors or the PI3K signaling pathway. (Xiv) In vivo drug combinations MOR10703
[0564] [00564] Since HER3 inhibition combined with target tyrosine kinase receptor agents in vitro, the impact of either MOR10701 or MOR10703 in combination with trastuzumab and erlotinib in vivo was evaluated. BT-474 in xenografts (see Figure 15A), the combination of either MOR10701 or MOR10703 (20mg / kg) with a sub-optimal dose of trastuzumab (1mg / kg) was sufficient to induce tumor regression (T / C % = -50 and -37, respectively). In L3.3 pancreatic xenografts, combination of MOR10703 (20mg / kg) with daily erlotinib (50mg / kg) resulted in tumor stasis (% T / C 15B = 3, see figure). In both models, the combination of the two drugs was significantly more effective than either drug alone, thus supporting our previous in vitro finding of the benefit of combining target HER3 antibodies with the target ErbB agents.
[0565] [00565] In summary, the unique ability of this family of antibodies to stabilize the inactive conformation of HER3 results in significant in vivo efficacy in models where HER3 is activated in either ligand in a dependent or independent manner. In addition, HER3 inhibition by this family of antibodies appears beneficial in combination with a wide variety of specific therapies. Incorporation by reference
[0566] [00566] All references in the present invention cited, including patents, patent applications, papers, books and the like, and in the references cited therein, insofar as they are no longer, are incorporated into the present invention by reference in their entirety. . Equivalents
[0567] [00567] The previous written specification is considered sufficient to allow one skilled in the art to practice the present invention. The foregoing description and examples in detail of certain preferred embodiments of the present invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the above may appear in the text, the present invention can be practiced in many ways, and the present invention must be interpreted in accordance with the appended claims and all their equivalents.

权利要求:
Claims (16)
[0001]
Isolated monoclonal antibody or antigen-binding fragment thereof to the HER3 receptor, characterized by the fact that it comprises sequences selected from the group consisting of a heavy chain variable region CDR1 of SEQ ID NO: 2; CDR2 of SEQ ID NO: 3; CDR3 of SEQ ID NO: 4; a light chain variable region CDR1 of SEQ ID NO: 5; CDR2 of SEQ ID NO: 6; and CDR3 of SEQ ID NO: 7; a heavy chain variable region CDR1 of SEQ ID NO: 20; CDR2 of SEQ ID NO: 21; CDR3 of SEQ ID NO: 22; a light chain variable region CDR1 of SEQ ID NO: 23; CDR2 of SEQ ID NO: 24; and CDR3 of SEQ ID NO: 25; a heavy chain variable region CDR1 of SEQ ID NO: 38; CDR2 of SEQ ID NO: 39; CDR3 of SEQ ID NO: 40; a light chain variable region CDR1 of SEQ ID NO: 41; CDR2 of SEQ ID NO: 42; and CDR3 of SEQ ID NO: 43; a heavy chain variable region CDR1 of SEQ ID NO: 56; CDR2 of SEQ ID NO: 57; CDR3 of SEQ ID NO: 58; a light chain variable region CDR1 of SEQ ID NO: 59; CDR2 of SEQ ID NO: 60; and CDR3 of SEQ ID NO: 61; a heavy chain variable region CDR1 of SEQ ID NO: 74; CDR2 of SEQ ID NO: 75; CDR3 of SEQ ID NO: 76; a light chain variable region CDR1 of SEQ ID NO: 77; CDR2 of SEQ ID NO: 78; and CDR3 of SEQ ID NO: 79; a heavy chain variable region CDR1 of SEQ ID NO: 92; CDR2 of SEQ ID NO: 93; CDR3 of SEQ ID NO: 94; a light chain variable region CDR1 of SEQ ID NO: 95; CDR2 of SEQ ID NO: 96; and CDR3 of SEQ ID NO: 97; a heavy chain variable region CDR1 of SEQ ID NO: 110; CDR2 of SEQ ID NO: 111; CDR3 of SEQ ID NO: 112; a light chain variable region CDR1 of SEQ ID NO: 113; CDR2 of SEQ ID NO: 114; and CDR3 of SEQ ID NO: 115; a heavy chain variable region CDR1 of SEQ ID NO: 128; CDR2 of SEQ ID NO: 129; CDR3 of SEQ ID NO: 130; a light chain variable region CDR1 of SEQ ID NO: 131; CDR2 of SEQ ID NO: 132; and CDR3 of SEQ ID NO: 133; a heavy chain variable region CDR1 of SEQ ID NO: 146; CDR2 of SEQ ID NO: 147; CDR3 of SEQ ID NO: 148; a light chain variable region CDR1 of SEQ ID NO: 149; CDR2 of SEQ ID NO: 150; and CDR3 of SEQ ID NO: 151; a heavy chain variable region CDR1 of SEQ ID NO: 164; CDR2 of SEQ ID NO: 165; CDR3 of SEQ ID NO: 166; a light chain variable region CDR1 of SEQ ID NO: 167; CDR2 of SEQ ID NO: 168; and CDR3 of SEQ ID NO: 169; a heavy chain variable region CDR1 of SEQ ID NO: 182; CDR2 of SEQ ID NO: 183; CDR3 of SEQ ID NO: 184; a light chain variable region CDR1 of SEQ ID NO: 185; CDR2 of SEQ ID NO: 186; and CDR3 of SEQ ID NO: 187; a heavy chain variable region CDR1 of SEQ ID NO: 200; CDR2 of SEQ ID NO: 201; CDR3 of SEQ ID NO: 202; a light chain variable region CDR1 of SEQ ID NO: 203; CDR2 of SEQ ID NO: 204; and CDR3 of SEQ ID NO: 205; a heavy chain variable region CDR1 of SEQ ID NO: 218; CDR2 of SEQ ID NO: 219; CDR3 of SEQ ID NO: 220; a light chain variable region CDR1 of SEQ ID NO: 221; CDR2 of SEQ ID NO: 222; and CDR3 of SEQ ID NO: 223; a heavy chain variable region CDR1 of SEQ ID NO: 236; CDR2 of SEQ ID NO: 237; CDR3 of SEQ ID NO: 238; a light chain variable region CDR1 of SEQ ID NO: 239; CDR2 of SEQ ID NO: 240; and CDR3 of SEQ ID NO: 241; a heavy chain variable region CDR1 of SEQ ID NO: 254; CDR2 of SEQ ID NO: 255; CDR3 of SEQ ID NO: 256; a light chain variable region CDR1 of SEQ ID NO: 257; CDR2 of SEQ ID NO: 258; and CDR3 of SEQ ID NO: 259; a heavy chain variable region CDR1 of SEQ ID NO: 272; CDR2 of SEQ ID NO: 273; CDR3 of SEQ ID NO: 274; a light chain variable region CDR1 of SEQ ID NO: 275; CDR2 of SEQ ID NO: 276; and CDR3 of SEQ ID NO: 277; a heavy chain variable region CDR1 of SEQ ID NO: 290; CDR2 of SEQ ID NO: 291; CDR3 of SEQ ID NO: 292; a light chain variable region CDR1 of SEQ ID NO: 293; CDR2 of SEQ ID NO: 294; and CDR3 of SEQ ID NO: 295; a heavy chain variable region CDR1 of SEQ ID NO: 308; CDR2 of SEQ ID NO: 309; CDR3 of SEQ ID NO: 310; a light chain variable region CDR1 of SEQ ID NO: 311; CDR2 of SEQ ID NO: 312; and CDR3 of SEQ ID NO: 313; a heavy chain variable region CDR1 of SEQ ID NO: 326; CDR2 of SEQ ID NO: 327; CDR3 of SEQ ID NO: 328; a light chain variable region CDR1 of SEQ ID NO: 329; CDR2 of SEQ ID NO: 330; and CDR3 of SEQ ID NO: 331; a heavy chain variable region CDR1 of SEQ ID NO: 344; CDR2 of SEQ ID NO: 345; CDR3 of SEQ ID NO: 346; a light chain variable region CDR1 of SEQ ID NO: 347; CDR2 of SEQ ID NO: 348; and CDR3 of SEQ ID NO: 349; and a heavy chain variable region CDR1 of SEQ ID NO: 362; CDR2 of SEQ ID NO: 363; CDR3 of SEQ ID NO: 364; a light chain variable region CDR1 of SEQ ID NO: 365; CDR2 of SEQ ID NO: 366; and CDR3 of SEQ ID NO: 367.
[0002]
Isolated monoclonal antibody or antigen-binding fragment thereof to the HER3 receptor, characterized by the fact that it comprises a variable region of heavy chain CDR1 of SEQ ID NO: 128; CDR2 of SEQ ID NO: 129; CDR3 of SEQ ID NO: 130; a light chain variable region CDR1 of SEQ ID NO: 131; CDR2 of SEQ ID NO: 132; and CDR3 of SEQ ID NO: 133.
[0003]
Isolated antibody or antigen-binding fragment thereof to the HER3 receptor, characterized by the fact that it comprises sequences selected from the group consisting of a VH comprising SEQ ID NO: 15 and a VL comprising SEQ ID NO: 14; a VH comprising SEQ ID NO: 33 and a VL comprising SEQ ID NO: 32; a VH comprising SEQ ID NO: 51 and a VL comprising SEQ ID NO: 50; a VH comprising SEQ ID NO: 69 and a VL comprising SEQ ID NO: 68; a VH comprising SEQ ID NO: 87 and a VL comprising SEQ ID NO: 86; a VH comprising SEQ ID NO: 105 and a VL comprising SEQ ID NO: 104; a VH comprising SEQ ID NO: 123 and a VL comprising SEQ ID NO: 122; a VH comprising SEQ ID NO: 141 and a VL comprising SEQ ID NO: 140; a VH comprising SEQ ID NO: 159 and a VL comprising SEQ ID NO: 158; a VH comprising SEQ ID NO: 177 and a VL comprising SEQ ID NO: 176; a VH comprising SEQ ID NO: 195 and a VL comprising SEQ ID NO: 194; a VH comprising SEQ ID NO: 213 and a VL comprising SEQ ID NO: 212; a VH comprising SEQ ID NO: 231 and a VL comprising SEQ ID NO: 230; a VH comprising SEQ ID NO: 249 and a VL comprising SEQ ID NO: 248; a VH comprising SEQ ID NO: 267 and a VL comprising SEQ ID NO: 266; a VH comprising SEQ ID NO: 285 and a VL comprising SEQ ID NO: 284; a VH comprising SEQ ID NO: 303 and a VL comprising SEQ ID NO: 302; a VH comprising SEQ ID NO: 321 and a VL comprising SEQ ID NO: 320; a VH comprising SEQ ID NO: 339 and a VL comprising SEQ ID NO: 338; a VH comprising SEQ ID NO: 357 and a VL comprising SEQ ID NO: 356; and a VH comprising SEQ ID NO: 375 and a VL comprising SEQ ID NO: 374.
[0004]
Isolated antibody or antigen-binding fragment thereof, characterized by the fact that it comprises a VH comprising SEQ ID NO: 141 and a VL comprising SEQ ID NO: 140.
[0005]
An isolated antibody or antigen-binding fragment thereof, characterized by the fact that it comprises a variable sequence of the heavy chain having SEQ ID NO: 493 and a variable sequence of the light chain having SEQ ID NO: 494.
[0006]
Isolated antigen-binding fragment according to any one of claims 1 to 5, characterized in that the fragment is selected from the group consisting of Fab, F (ab2) 'and scFv.
[0007]
Pharmaceutical composition for the treatment of cancer, characterized in that it comprises an antibody or its antigen-binding fragment, as defined in any one of claims 1 to 6, and a pharmaceutically acceptable carrier.
[0008]
Pharmaceutical composition according to claim 7, characterized by the fact that it further comprises an additional therapeutic agent.
[0009]
Pharmaceutical composition according to claim 8, characterized by the fact that the additional therapeutic agent is an HER1 inhibitor selected from the group consisting of Matuzumab (EMD72000), Erbitux® / Cetuximab, Vectibix® / Panitumumab, mAb 806, Nimotuzumab, Iressa® / Gefitinib, CI-1033 (PD183805), Lapatinib (GW-572016), Tykerb® / Lapatinib Ditosylate, Tarceva® / Erlotinib HCL (OSI-774), PKI-166 and Tovok®; an HER2 inhibitor selected from the group consisting of Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib or lapitosib or lapatinib / Tykerb® ditosylate; an HER3 inhibitor selected from the group consisting of, MM-121, MM-111, IB4C3, 2DID12 (U3 Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech) and small molecules that inhibit HER3 ; or an HER4 inhibitor.
[0010]
Pharmaceutical composition according to claim 8, characterized by the fact that the additional therapeutic agent is an HER2 inhibitor.
[0011]
Pharmaceutical composition according to claim 10, characterized by the fact that the additional therapeutic agent is an HER2 inhibitor selected from the group consisting of Pertuzumab, Trastuzumab, MM-111, neratinib, lapatinib or lapatinib ditosylate.
[0012]
Pharmaceutical composition according to claim 8, characterized by the fact that the additional therapeutic agent is an mTOR inhibitor selected from the group consisting of Temsirolimus, ridaforolimus and everolimus.
[0013]
Pharmaceutical composition according to claim 8, characterized by the fact that the additional therapeutic agent is an inhibitor of PI3 kinase selected from the group consisting of GDC 0941, BEZ235, BMK120 and BYL719.
[0014]
Pharmaceutical composition, characterized by the fact that it is for the treatment of cancer mediated by an HER3 ligand-dependent signal transduction or ligand-independent signal transduction path selected from the group consisting of breast cancer, colorectal cancer, cancer of lung, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, cancer of head and neck, bladder cancer, esophageal cancer, glioblastoma, soft tissue sarcoma of soft tissues, malignant mesothelioma, neurofibromatosis, kidney cancer and melanoma, comprising an antibody or its antigen binding fragment, as defined in any of the claims 1-5.
[0015]
Pharmaceutical composition for the treatment of cancer, characterized by the fact that it comprises an antibody or its antigen-binding fragment with VH of SEQ ID NO: 141 and VL of SEQ ID NO: 140.
[0016]
Use of an antibody or its antigen-binding fragment, as defined in any of claims 1-5, or of a pharmaceutical composition, as defined in any of claims 7 to 15, characterized by the fact that it is for the manufacture of a medicine for the treatment of cancer mediated by an HER3 ligand-dependent signal transduction or ligand-independent signal transduction pathway selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer , liver cancer, gastric cancer, pancreatic cancer, prostate cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, glioblastoma, soft tissue sarcoma, soft tissue, malignant mesothelioma, neurofibromatosis, kidney cancer and melanoma.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-13| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-27| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-23| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/08/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US37540810P| true| 2010-08-20|2010-08-20|

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US61/375,408|2010-08-20|

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PCT/EP2011/064407|WO2012022814A1|2010-08-20|2011-08-22|Antibodies for epidermal growth factor receptor 3 |

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