HK1173158B

HK1173158B - Antibodies directed to her-3 and uses thereof

Info

Publication number: HK1173158B
Application number: HK13100462.4A
Authority: HK
Inventors: M．罗瑟; M．特里德; S.哈特曼; D.弗里曼; B．拉丁斯基; E.伯吉斯
Original assignee: 第一三共欧洲有限公司; 安进公司
Priority date: 2005-12-30
Filing date: 2013-01-11
Publication date: 2015-07-31

Description

Antibodies to HER-3 and uses thereof

The application is a divisional application of a patent with the same name and with the application number of 200680049887.7 and the application date of 2006, 12 and 29.

Technical Field

The present invention relates to binding proteins, including antibodies and binding fragments thereof that bind to HER-3 and polynucleotide sequences encoding the same. The invention also provides expression vectors and host cells comprising the expression vectors for producing the binding proteins of the invention. In addition, the invention provides compositions and methods for diagnosing and treating diseases associated with HER-3 mediated signal transduction and/or its ligand regulatory protein (heregulin).

Technical Field

Human epidermal growth factor receptor 3(HER-3, also known as ErbB3) is a receptor protein tyrosine kinase belonging to the Epidermal Growth Factor Receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER-1 (also known as EGFR), HER-2 and HER-4(Plowman et al Proc. Natl. Acad. Sci. U.S.A.87(1990), 4905-4909; Kraus et al Proc. Natl. Acad. Sci.U.S.A.86(1989), 9193-9197; and Kraus et al Proc. Natl. Acad. Sci.U.S.A.90(1993), 2900-2904). Like the classical epidermal growth factor receptor, the transmembrane receptor HER-3 consists of an extracellular ligand binding domain (ECD), a dimeric domain within the ECD, a transmembrane domain, an intracellular protein Tyrosine Kinase Domain (TKD), and a C-terminal phosphorylation domain.

Ligand regulin (HRG) binds to the extracellular domain of HER-3 and activates receptor-mediated signaling pathways by promoting dimerization with other human epidermal growth factor receptor (HER) family members and phosphorylation of the transmembrane domain of HER-3. Dimers among the HER family members amplify the signal potential of HER-3, both in a signal-diverse and signal-amplified fashion. For example, of the HER family members, the HER-2/HER-3 heterodimer induces one of the most important mitogenic signals.

HER-3 has been found to be overexpressed in some types of cancer, such as breast, gastrointestinal and pancreatic cancer. Interestingly, studies have shown a correlation between HER-2/HER-3 expression and Cancer progression from the non-invasive to the invasive stage (Oncogene 10, 1813-1821, by Alinandi et al; Cancer 87, 487-498, by deFazio et al; Br. J. Cancer 78, 1385-1390, by Naidu et al). Accordingly, agents that interfere with HER-3 mediated signal transduction are desirable. Murine or chimeric HER-3 antibodies have been reported, for example in patents US 5968511, US 5480968 and WO 03013602.

Recent studies have shown that a humanized monoclonal antibody directed against HER-2 is HEISEPIInterfere with HER-2 mediated signal transduction and have therapeutic effects on human cancers (hybrid 6, 359-370, Fendly et al, MoI, cell. biol.9, 1165-1172, Hudziak et al, Cancer treat. Rev.26, 287-290). Has shown that HersaiCan act by two different mechanisms, namely the involvement of effector cells of the immune system and direct cytotoxicity andinduction of apoptosis.

However, only those patients with high HER-2 expression will be refractory to HisexIs significant, thus limiting the number of patients eligible for treatment. Furthermore, the development of resistance or changes in HER-2 expression or its epitope sequence in tumor cells may render those patients who would be expected to be treated unable to react with the antibody and thereby abrogate its therapeutic effect. Thus, there is a need for more targeted therapeutic agents that are directed to other members of the HER family, such as HER-3.

Brief description of the drawings

FIG. 1 shows the extent of HER-3 expression in a range of human cancer cell lines, indicating that HER-3 is expressed in a variety of human cancers.

FIG. 2 shows FACS analysis of the binding of HER-3 antibodies to Rat1 cells stably expressing different members of the HER family or Rat1 cells containing only the empty vector.

Figure 3 shows that antibody competing bins are localized to the HER3 domain.

FIG. 4 shows the results of an indirect FACSSScatchard antibody affinity assay using the anti-HER-3 antibody of the invention. The analysis shows that the anti-HER-3 antibody of the invention has high affinity and high binding constant for HER-3 expressed on the cell surface.

FIG. 5 shows that the anti-HER-3 antibodies of the invention accelerate the endocytosis of HER-3.

FIGS. 6a-e show the results of a ligand competition assay using an anti-HER-3 antibody of the invention. These results indicate that the antibodies of the invention specifically reduce [125I ]]-α-HRG/[¹²⁵I]Binding of β -HRG to cells expressing endogenous HER-3.

FIG. 7a shows the results of a HER-3 phosphotyrosine enzyme-linked immunosorbent assay (ELISA) using the anti-HER-3 antibody of the invention. The antibodies of the invention are capable of inhibiting beta-HRG-mediated HER-3 activation as evidenced by an increase in the degree of receptor tyrosine phosphorylation. Figure 7b shows representative results of this experiment using titrated antibodies.

FIG. 8 shows the results of an enzyme-linked immunosorbent assay using the anti-HER-3 antibody of the invention and p42/p44MAP kinase. The antibodies of the invention are capable of reducing beta-HRG mediated activation of p42/p44 MAP-kinase, as evidenced by an increase in the degree of phosphorylation of MAP-kinase.

FIG. 9 shows the results of an ELISA assay using the anti-HER-3 antibody of the invention and phosphoAKT. The antibodies of the invention are capable of reducing beta-HRG mediated AKT activation, manifested as phosphorylation of AKT.

FIG. 10 shows that the human anti-HER-3 antibody of the invention inhibits the proliferation of MCF7 cells. The antibodies of the invention inhibit HRG-induced cell proliferation in human cancer cells.

FIG. 11 shows that the human anti-HER-3 antibody of the invention inhibits the transfer of MCF7 cells.

FIGS. 12a-i show that human anti-HER-3 antibodies of the invention inhibit anchorage-independent cell growth.

FIG. 13 shows the inhibition of xenograft growth of T47D human breast cancer cells by human anti-HER-3 antibodies of the invention.

FIG. 14 shows that the number of BxPC3 human pancreatic cancer cells in mice was reduced by administering anti-Her 3(U1-59and U1-53) or anti-EGFR (erbitux) antibodies to the mice.

FIG. 15 shows that human anti-HER-3 antibodies of the invention, in combination with anti-EGFR (erbitux) antibodies, reduced xenograft growth of BxPC3 human pancreatic cancer cells.

FIG. 16 shows that the antibodies of the invention retard the growth of melanoma cells (HT144) in nu/nu mice.

FIG. 17 shows that human HER-3 antibodies of the invention (U1-53, U1-59and U1-7) reduced xenograft growth of human colon cancer cells HT-29.

FIG. 18 shows that the human anti-HER-3 antibody of the invention (U1-59, U1-53and U1-7) reduced xenograft growth of human lung cancer cell Calu-3.

FIG. 19 shows that human anti-HER-3 antibodies of the invention (U1-7, U1-59and U1-53) reduced xenograft growth of human pancreatic cancer cells BxPC 3.

FIG. 20 shows that the antibody of the invention (U1-59) causes HER-3 inhibition in BxPC3 human pancreatic cancer xenografts.

Summary of The Invention

The first aspect of the present invention relates to an isolated binding protein that binds to HER-3.

In one embodiment of the invention, an isolated binding protein of the invention comprises:

a heavy chain amino acid sequence comprising at least one Complementarity Determining Region (CDR) selected from the group consisting of seq id nos:

(a) SEQ ID NOs: 2. CDRH1 shown at 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, and 230;

(b) SEQ ID NOs: 2. CDRH2 shown at 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, and 230; and

(c) SEQ ID NOs: 2. CDRH3 shown at 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, and 230; and/or

A light chain amino acid sequence comprising at least one Complementarity Determining Region (CDR) selected from the group consisting of seq id nos:

(d) SEQ ID NOs: 4. CDRL1 shown in 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, and 232;

(e) SEQ ID NOs: 4. CDRL2 shown in 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, and 232; and

(f) SEQ ID NOs: 4. CDRL3 shown in 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, and 232.

In another embodiment of the invention, an isolated binding protein of the invention comprises:

a heavy chain amino acid sequence selected from the group consisting of seq id no:

SEQ ID Nos: 2. 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, and 230; and/or

A light chain amino acid sequence selected from the group consisting of seq id no:

SEQ ID Nos: 4. 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, and 232.

the heavy and light chain amino acid sequences shown below:

SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, 22 and 24, 26 and 28, 30 and 32, 36 and 38, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 60 and 58, 62 and 64, 66 and 68, 70 and 72, 74 and 76, 78 and 82, 80 and 82, 84 and 86, 88 and 90, 92 and 94, 96 and 98, 100 and 102, 104 and 106, 108 and 110, 112 and 114, 116 and 118, 122 and 124, 126 and 128, 130 and 132, 134 and 136, 138 and 140, 142 and 144, 146 and 148, 150 and 152, 154 and 156, 158 and 160, 162 and 164, 166 and 168, 170 and 172, 174 and 176, 178 and 180, 182 and 184, 186 and 188, 190 and 192, 194 and 196, 198 and 200, 202 and 204, 206 and 208, 210 and 212, 214 and 216, 218 and 220, 222 and 226 and 232, 222 and 232; or

SEQ ID NOs: 34. 40, 60, 62 or 120; or

SEQ ID NOs: 58 or 64.

According to the invention, an isolated binding protein capable of binding to HER-3 interacts with at least one epitope of the extracellular part of HER-3. These epitopes are preferably located in the L1 domain (amino acids 19-184), the S1 (amino acids 185-327) and the S2 domain (amino acids 500-632) or the L2 domain (amino acids 328-499), wherein the L1 domain is the amino-terminal domain, the S1 and S2 domains are the two cysteine-rich domains, and the L2 is flanked by the two cysteine-rich domains. These epitopes can also be located in a combination of domains, but are not limited to epitopes consisting of parts of L1 and S1. Even more preferred is an isolated binding protein capable of binding to a three-dimensional structure formed by amino acid residues 1-160, 161-358, 359-575, 1-358 and/or 359-604 of mature HER-3, particularly mature human HER-3 protein.

Preferably, an isolated binding protein of the invention is a scaffold protein, has antibody-like binding activity or is an antibody, such as an antibody against HER-3. In particular, the anti-HER-3 antibody is selected from the group consisting of:

u1-1 antibody, U1-2 antibody, U1-3 antibody, U1-4 antibody, U1-5 antibody, U1-6 antibody, U1-7 antibody, U1-8 antibody, U1-9 antibody, U1-10 antibody, U1-11 antibody, U1-12 antibody, U1-13 antibody, U1-14 antibody, U1-15 antibody, U1-16 antibody, U1-17 antibody, U1-18 antibody, U1-19 antibody, U1-20 antibody, U1-21 antibody, U1-22 antibody, U1-23 antibody, U1-24 antibody, U1-25 antibody, U1-26 antibody, U1-27 antibody, U1-28 antibody, U1-29 antibody, U1-30 antibody, U1-31 antibody, U1-32 antibody, U1-33 antibody, U1-34 antibody, U1-35 antibody, U1-36 antibody, U1-37 antibody, U1-38 antibody, U1-39 antibody, U1-40 antibody, U1-41 antibody, U1-42 antibody, U1-43 antibody, U1-44 antibody, U1-45 antibody, U1-46 antibody, U1-47 antibody, U1-48 antibody, u1-49 antibody, U1-50 antibody, U1-51 antibody, U1-52 antibody, U1-53 antibody, U1-55.1 antibody, U1-55 antibody, U1-57.1 antibody, U1-57 antibody, U1-58 antibody, U1-59 antibody, U1-61.1 antibody, U1-61 antibody, U1-62 antibody or an antibody having at least one heavy chain or light chain of one of the antibodies. Particularly preferred are the U1-49 antibody (SEQ ID NO: 42/44), the U1-53 antibody (SEQ ID NO: 54/56) and the U1-59 antibody (SEQ ID NO: 70/72) antibodies or antibodies with at least one of the heavy or light chains of one of the antibodies.

In addition, additional embodiments of the invention provide isolated binding proteins conjugated to a labeling group or an effector group. Preferably, such binding proteins are useful for the treatment of hyperproliferative diseases, in particular neoplastic diseases, such as: breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, testicular cancer, soft tissue sarcoma, head and neck cancer, other cancers that express or overexpress HER-3, and the formation of tumor metastases.

Other aspects of the invention relate to isolated nucleic acid molecules encoding binding proteins of the invention, vectors containing isolated nucleic acid molecules encoding binding proteins of the invention, and a host cell, such as: CHO cells and NS/0 myeloma cells are transformed with the nucleic acid molecule or vector.

Another aspect of the invention relates to a method of producing a binding protein of the invention by producing said binding protein from a host cell that secretes the binding protein. Preferably, the binding proteins of the invention are prepared from hybridoma cell lines secreting the binding protein or from CHO cells or other cell types transformed with a nucleic acid molecule encoding the binding protein of the invention.

Another aspect of the invention relates to a method for producing a binding protein of the invention from a tissue, product or secretion of a transgenic animal, transgenic plant or fungus directed against one or more nucleic acid molecules encoding a binding protein of the invention. Preferably, the binding proteins of the invention are prepared from tissues, products or secretions of transgenic animals such as cows, sheep, rabbits, chickens or other mammals or birds, transgenic plants such as maize, tobacco or other plants, or transgenic fungi such as aspergillus, pichia or other fungal species.

Another aspect of the invention relates to a pharmaceutical composition comprising as an active agent at least one binding protein of the invention in admixture with a pharmaceutically acceptable carrier, diluent and/or adjuvant. In another preferred embodiment of the present invention, the pharmaceutical composition of the present invention additionally comprises at least one further active agent, such as: at least one antineoplastic agent. Other aspects of the invention also relate to the use of at least one binding protein of the invention, optionally at least one other active agent, such as at least one antineoplastic agent, in admixture with a pharmaceutically acceptable carrier, diluent and/or adjuvant, for the preparation of a pharmaceutical composition. Such pharmaceutical compositions are suitable for the diagnosis, prevention or treatment of a hyperproliferative disease, in particular a neoplastic disease, such as breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, renal cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, or other HER-3 expressing or overexpressing cancer and the formation of tumor metastases.

In addition, another aspect of the invention relates to a method for diagnosing a disease or condition associated with HER-3 expression, comprising contacting a sample with a composition comprising at least one binding protein of the invention, and detecting the presence of HER-3. Preferred diseases or conditions include the hyperproliferative diseases mentioned above.

Another aspect of the invention is a method of preventing or treating a disease or condition associated with HER-3 expression in a patient in need thereof, comprising administering to the patient an effective amount of at least one binding protein of the invention, and optionally at least one other active agent, such as at least one anti-neoplastic agent. Preferably the patient is a mammalian patient, more preferably a human patient. Preferred diseases or conditions associated with HER-3 expression are the hyperproliferative diseases mentioned above.

Another aspect of the invention relates to a kit for the diagnosis, prevention or treatment of a disease or condition associated with HER-3 expression, comprising at least one binding protein and/or nucleic acid molecule and/or vector of the invention. Optionally, the kits of the present invention may further comprise at least one additional active agent, such as at least one antineoplastic agent. Preferably, the disease or condition associated with HER-3 expression is a hyperproliferative disease as mentioned above.

Detailed Description

the heavy and light chain amino acid sequences shown below:

SEQ ID NOs: 34. 40, 60, 62 or 120; or

SEQ ID NOs: 58 or 64.

According to the present invention, it is to be understood that the amino acids of the binding proteins of the invention are not limited to only 20 common amino acids (see Immunology-A Synthesis (second edition, E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference). For example, an amino acid molecule may include stereoisomers of 20 conventional amino acids (e.g., D-form amino acids), unnatural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unusual amino acids. Unusual amino acids may also be suitable components of the binding proteins of the invention, examples of which include: 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino acid based imino acids, such as 4-hydroxyproline.

Furthermore, according to the invention, if the variation is maintained in SEQ ID NOs: 1-232, more preferably at least 80%, 90%, 95%, most preferably 99%, such that the amino acid sequence set forth in SEQ ID NOs: 1-232 are also contemplated to be encompassed by the present invention. These variations may occur in the framework region (i.e., outside the complementarity determining region), in the complementarity determining region, or both the framework region and the complementarity determining region. SEQ ID NOs: 1-232, i.e., deletion, insertion and/or substitution of at least one amino acid, occurs near the boundaries of the functional domains. Structural and functional domains can be identified by alignment of nucleic acid and/or amino acid data with public or proprietary sequence databases. Computer alignment methods can be used to discriminate sequence motifs or predict the presence of protein conformation domains of other binding proteins of known structure and/or function. Methods for discriminating protein sequences that fold into a known three-dimensional structure are known. Reference is made to Bowie et al Science 253, 164(1991), Proteins, Structures and Molecular Principles (Creighton, Ed., W.H.Freeman and Company, New York (1984)), Introduction to Protein Structure (C.Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)), and Thornton et al Nature 354, 105(1991), which are incorporated herein by reference. Thus, in accordance with the present invention, one skilled in the art is able to recognize sequence motifs and structural conformations that may be used to define structural and functional domains.

SEQ ID NOs: 1-174 and 1-232 are those that result in a binding protein that has reduced susceptibility to proteolysis or oxidation, altered glycosylation patterns or altered binding affinity or imparts or modifies other physicochemical or functional properties. In particular, conservative amino acid substitutions are also contemplated. Conservative substitutions occur within a family of amino acids that are related to the side chain. Preferred amino acid families are as follows: the acidic family is aspartic acid, glutamic acid; basic family ═ lysine, arginine, histidine; non-polar family ═ alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and the family of uncharged polar amino acids-glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred amino acid families are: the aliphatic hydroxyl family ═ serine and threonine; the family of amides, asparagine and glutamine; the aliphatic family ═ alanine, valine, leucine, and isoleucine; and the aromatic amino acids phenylalanine, tryptophan and tyrosine. For example, it is reasonably predictable that the replacement of leucine with isoleucine or valine, of aspartic acid with glutamic acid, of threonine with serine alone; or a similar substitution of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the binding protein, particularly when the substitution does not include amino acids at the framework regions. However, all other possible amino acid substitutions are also contemplated by the present invention. Whether an amino acid change results in a functional binding protein, that is, a binding protein that binds to HER-3 and reduces signal transduction of HER family members, can be readily determined by enzyme-linked immunosorbent assay (ELISA) or FACS analysis of the specific activity of the resulting protein to bind to HER-3 or in vivo or in vitro functional assays.

According to the invention, the binding protein of the invention interacts with at least one epitope of the extracellular part of HER-3. These epitopes are preferably located in the L1 domain (amino acids 19-184), the S1 (amino acids 185-327) and S2 domains (amino acids 500-632) or the L2 domain (amino acids 328-499), or in a combination of HER-3 domains, where the L1 domain is the amino-terminal domain and the S1 and S2 domains are two cysteine-rich domains flanking the L2 domain. These epitopes can also be located in a combination of domains, but are not limited to epitopes consisting of parts of L1 and S1. Furthermore, the binding proteins of the invention are further characterized in that they are capable of binding to HER-3 and reduce HER-3 mediated signal transduction. According to the invention, a reduction of HER-3 mediated signal transduction may, for example, be due to down-regulation of HER-3, resulting in at least partial disappearance of the HER-3 molecule from the cell surface; or because HER-3 is essentially stable on the cell surface in an inactive form, i.e.a form which shows lower signal transduction compared to the non-stable form. Alternatively, the reduction in HER-3 mediated signal transduction may also be due to affecting (e.g., reducing or inhibiting) binding of a ligand or another member of the HER family to HER-3 and binding of GRB2 to HER-2 or SHC by inhibiting receptor tyrosine phosphorylation, AKT phosphorylation, PYK2 tyrosine phosphorylation or ERK2 phosphorylation, or by reducing tumor invasiveness. Alternatively, the reduction in HER-3 mediated signal transduction may also be due to an effect (e.g., reduction or inhibition) of HER-3 dimer formation with other HER family members. One of the other embodiments may be that the formation of a HER-3-EGFR protein complex is reduced or inhibited.

Preferably, the binding protein of the invention is a scaffold protein, having antibody-like binding activity or is an antibody, i.e. an antibody against HER-3.

The term "scaffold protein" as used in the context of the present invention refers to a polypeptide chain or protein containing exposed surface regions which is highly tolerant to amino-terminal insertions, substitutions or deletions. Examples of scaffold proteins which can be used according to the invention are protein A of Staphylococcus aureus, the bilin-binding protein of Pieris brassicae, or other lipocalins, ankyrin repeat proteins and human fibronectin (for a review: Binz and Pluckthun, Curr Opin Biotechnol, 16, 459-69). Engineering of scaffold proteins can be viewed as grafting or integrating affinity functions onto or into the structural framework that stabilizes the folded protein. According to the present invention, affinity function refers to protein binding affinity. The scaffold may be structurally independent of the amino acid sequence conferring the binding property. In general, proteins that appear to be suitable for the development of such artificial affinity reagents can be used for in vitro display of artificial scaffold libraries as binding reagents by rational, or most common, combinatorial protein engineering techniques such as screening for HER-3, using purified proteins or proteins displayed on the cell surface, as are well known in the art (Skerra, J.mol.Recog., 2000; Binz and Pl ü ckthun, 2005). Furthermore, a scaffold protein with antibody-like binding activity may be derived from an acceptor polypeptide comprising the scaffold domain, which may be grafted with the binding domain of the acceptor polypeptide and confer the binding properties of a donor polypeptide to an acceptor polypeptide comprising such a scaffold domain. The intervening binding domain may be, for example, a Complementarity Determining Region (CDR) of an antibody, particularly a CDR of an anti-HER-3 antibody. Insertion can be accomplished by a variety of art-recognized methods, for example, polypeptide synthesis, nucleic acid synthesis encoding amino acids, and by a variety of recombinant methods well known to those skilled in the art.

Furthermore, the term "antibody" or "anti-HER-3 antibody" as used herein refers to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody (Jones et al, Nature321(1986), 522-525; Riechmann et al, Nature 332(1988), 323-329; and Presta, curr. Op. struct. biol.2(1992), 593-596), a chimeric antibody (Morrison et al, Proc. Natl.Acad.Sci.U.S.A.81(1984), 6851-6855), a multispecific antibody (e.g., a diabody) consisting of at least two antibodies, or an antibody fragment thereof. The term "antibody fragment" includes any part of the above mentioned antibodies, preferably their antigen binding or variable regions. Examples of antibody fragments include Fab fragments, Fab 'fragments, F (ab')₂Fragment, Fv fragment, diabody (Hollinger)Et al, proc.Natl.Acad.Sci.U.S.A.90(1993), 6444-6448), single chain antibody molecules (Plu u ckthun: the Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore, EDS, Springer Verlag, N.Y. (1994), 269-315) and other fragments, as long as The desired HER-3 binding capacity is exhibited.

Furthermore, the term "antibody" or "anti-HER-3 antibody" as used herein may encompass antibody-like molecules including engineered antibody subdomains or naturally occurring antibody variants. These antibody-like molecules may be single domain antibodies such as single VH or VL domains, either of natural origin such as camelids (Muydermans et al, Reviews in molecular Biotechnology 74, 277-302), or obtained by human, camelids or other species in vitro display libraries (Holt et al, Trends Biotechnology, 21, 484-90).

According to the invention, an "Fv fragment" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy or light chain formed by tight non-covalent association. It is in this configuration that the three CDRs of each variable region interact to define V_H-V_LSurface antigen binding sites of dimers. In general, these six complementarity determining regions confer the antigen binding properties of the antibody. However, even a single variable domain (or half of an Fv fragment comprising only three complementarity determining regions specific for an antigen) has the ability to recognize and bind antigen, although generally with lower affinity than the entire binding site. The "Fab fragment" also includes the constant region of the light chain and the first constant region of the heavy chain (CH 1). A "Fab fragment" is distinguished from a "Fab' fragment" in that residues are added to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. "F (ab')₂"originally produced from a pair of" Fab' fragments "which contain a hinge cysteine in between. Methods for preparing such antibody fragments, such as papain or pepsin digestion, are well known to those skilled in the art.

In a preferred embodiment of the invention, the anti-HER-3 antibody of the invention is of the IgA-, IgD-, IgE-, IgG-or IgM-type, preferably of the IgG-or IgM-type, including, but not limited to, the IgG1-, IgG2-, IgG3-, IgG4-, IgM 1-and IgM 2-type. In the most preferred embodiment, the antibody is of the IgG1-, IgG2-, or IgG 4-type.

In another preferred embodiment of the invention, the anti-HER-3 antibody of the invention is an anti-HER-3 antibody raised against the extracellular domain (ECD) of HER-3.

In certain aspects, e.g., in connection with the production of anti-HER-3 antibodies as therapeutic candidates, it is desirable that the anti-HER-3 antibodies of the invention are capable of fixing complement and participate in complement-dependent cytotoxicity (CDC). Many antibody isotypes can be identical, including (but not limited to) the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, human lgG3, and human IgA. It will be appreciated that the antibody produced need not initially have such an isotype, but rather, the antibody produced may have any isotype, such an antibody may be a converted isotype by appending the molecularly cloned V region gene or cDNA to the molecularly cloned constant region gene or cDNA molecule in a suitable expression vector using conventional biological techniques well known in the art, and then expressing the antibody in a host cell by techniques well known in the art. Such isotype-switched antibodies may also have an Fc region that has been engineered to have better complement dependent cytotoxicity than naturally occurring variants (Idusogene et al, JImmunol., 166, 2571-2575) and may be recombinantly expressed in host cells by techniques well known in the art. These techniques include the use of direct recombinant techniques (see U.S. Pat. No.4,81(5,397), cell fusion techniques (see U.S. Pat. Nos.5,916,771 and 6,207,418), and the like, among the cell fusion techniques, a myeloma or other cell line such as CHO is prepared having any desired isotype of heavy chain, other myeloma cells or other cell lines such as CHO are prepared having light chains, these cells can then be fused and the cell line expressing the intact antibody can be isolated, as an example, a human anti-HER-3 IgG4 antibody, having the desired ability to bind to the HER-3 antigen, human IgM, human IgG1 or IgG3 isotypes can be generated by a simple isotype switch, while still having the same variable regions (which define the specificity and some affinity of the antibody) such molecules may then be able to fix complement and participate in complement-dependent cytotoxicity.

Furthermore, it is also desirable that the anti-HER-3 antibodies of the invention are capable of binding to Fc receptors of effector cells, such as monocytes and natural killer cells (NK), and participate in antibody-dependent cellular cytotoxicity (ADCC). There are many isoforms of antibodies that can perform the same function, including (but not limited to) the following: murine IgG2a, murine IgG2b, murine IgG3, human IgG1, and human IgG 3. It will be appreciated that the antibody produced need not initially have such an isotype, but rather, the antibody produced may have any isotype, such an antibody may be of the converted isotype by appending the V region gene or cDNA of the molecular clone to the constant region gene or cDNA molecule of the molecular clone in a suitable expression vector using conventional biological techniques well known in the art, and then expressing the antibody in a host cell by techniques well known in the art. Such isotype-switched antibodies may also have an Fc region that has been engineered to have better antibody-dependent cytotoxicity than naturally occurring variants (Shields et al, J Biol chem., 276, 6591-6604), and may be recombinantly expressed in host cells by techniques well known in the art. These techniques include the use of direct recombinant techniques (see U.S. Pat. No.4,816,397), cell fusion techniques (see U.S. Pat. Nos.5,916,771 and 6,207,418), and the like. In cell fusion techniques, a myeloma or other cell line such as CHO is prepared having heavy chains of any desired isotype, and other myeloma cells or other cell lines such as CHO are prepared having light chains. These cells can then be fused and cell lines expressing intact antibodies can be isolated. As an example, a human anti-HER-3 IgG4 antibody, with the desired ability to bind to the HER-3 antigen, can be used to generate human IgG1 or IgG3 isotypes by simple isotype switching, while still having the same variable regions (which define the specificity and some affinity of the antibody). Such molecules may then be able to bind to Fc γ R of effector cells and be able to participate in antibody-dependent cytotoxicity.

Furthermore, it will be appreciated that, in accordance with the present invention, an anti-HER-3 antibody of the invention is a fully human or humanized antibody. Human antibodies avoid certain problems caused by xenogenous antibodies (e.g., antibodies having variable and/or constant regions of murine or rat origin). Proteins of xenogenic origin, such as those of murine or rat origin, can cause the patient to develop an immune response against the antibody, which in turn leads to rapid clearance of the antibody, loss of therapeutic effect, by neutralization of the antibody and/or severe, even life-threatening, allergic reactions.

Preferably, the anti-HER-3 antibody of the invention is selected from the group consisting of:

u1-1 antibody, U1-2 antibody, U1-3 antibody, U1-4 antibody, U1-5 antibody, U1-6 antibody, U1-7 antibody, U1-8 antibody, U1-9 antibody, U1-10 antibody, U1-11 antibody, U1-12 antibody, U1-13 antibody, U1-14 antibody, U1-15 antibody, U1-16 antibody, U1-17 antibody, U1-18 antibody, U1-19 antibody, U1-20 antibody, U1-21 antibody, U1-22 antibody, U1-23 antibody, U1-24 antibody, U1-25 antibody, U1-26 antibody, U1-27 antibody, U1-28 antibody, U1-29 antibody, U1-30 antibody, U1-31 antibody, U1-32 antibody, U1-33 antibody, U1-34 antibody, U1-35 antibody, U1-36 antibody, U1-37 antibody, U1-38 antibody, U1-39 antibody, U1-40 antibody, U1-41 antibody, U1-42 antibody, U1-43 antibody, U1-44 antibody, U1-45 antibody, U1-46 antibody, U1-47 antibody, U1-48 antibody, U1-49 antibody, U1-50 antibody, U1-51 antibody, U1-52 antibody, U1-53 antibody, U1-55.1 antibody, U1-55 antibody, U1-57.1 antibody, U1-57 antibody, U1-58 antibody, U1-59 antibody, U1-61.1 antibody, U1-61 antibody, U1-62 antibody.

In a preferred embodiment of the invention, the binding protein of the invention is conjugated to a labelling group. Such binding proteins are particularly suitable for diagnostic applications. The term "labeling group" as used herein refers to a detectable label, such as a radiolabeled amino acid or biotinylated moiety, which can be labeled with an antibodyBiotin proteins (e.g.streptavidin conjugated with a fluorescent label or enzyme activity detectable using optical or colorimetric methods). Various methods known to those skilled in the art for labeling polypeptides or glycoproteins, such as antibodies, can be used to carry out the present invention. Examples of suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N，、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Fluorescent group (for example: FITC, rhodamine, lanthanide phosphors), enzyme groups (e.g.: horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), a chemiluminescent group, a biotin group, or a predetermined polypeptide epitope recognized by a second reporter (e.g.: leucine zipper pair sequence, binding site of second antibody, metal binding domain, epitope tag). In certain embodiments, it may be desirable for the labeling groups to be attached using spacer arms of different lengths to reduce potential steric hindrance.

Alternatively, in another preferred embodiment of the invention, the binding protein of the invention may be coupled to an effector group. Such binding proteins are particularly suitable for therapeutic applications. The term "effector group" as used herein refers to a cytotoxic group such as a radioisotope or radionuclide, a toxin, a therapeutic group, or other effector groups well known in the art. Examples of suitable effector groups are radioisotopes or radionuclides (e.g. as³H、¹⁴C、¹⁵N，、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) Calicheamicin, dolastatin analogs such as auristatins, and chemotherapeutic agents such as geldanamycin and maytansine derivatives, including DM 1. In certain embodiments, it may be desirable for the effector groups to be attached using spacer arms of different lengths to reduce potential steric hindrance.

A secondary aspect of the invention relates to a process for preparing an isolated binding protein of the invention, comprising the step of preparing the binding protein from a host cell that secretes the binding protein. Host cells that can be used according to the invention are hybridoma cells; eukaryotic cells such as mammalian cells, e.g., hamster, rabbit, rat, pig, mouse, or other animal cells; a plant cell; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris; prokaryotic cells such as E.coli; or other cells well known in the art, for the production of binding proteins. Various methods for preparing and isolating binding proteins (e.g., scaffold proteins or antibodies) from host cells are well known in the art and can be used to practice the present invention. Furthermore, methods for preparing binding protein fragments (such as scaffold protein fragments or antibody fragments), for example: papain or pepsin digestion, modern cloning techniques, techniques for The preparation of single-chain antibody molecules (see Pl ü ckthun: The Pharmacology of monoclonal Antibodies 113, Rosenburg and Moore, EDS, Springer Verlag, N.Y. (1994), 269-315) and diabodies (Hollinger et al, Proc. Natl. Acad. Sci. U.S. A.90(1993), 6444-6448), which are well known to The person skilled in The art, can be used to carry out The invention.

In a preferred embodiment of the invention, the binding protein of the invention is prepared from hybridoma cells that secrete the binding protein. See K ǒ hler et al, Nature 256(1975), 495.

In a more preferred embodiment of the invention, the binding protein of the invention is produced recombinantly, wherein the binding protein is expressed optimally or/and amplified in a host cell and isolated from said cell. For this purpose, host cells are transformed or transfected with DNA encoding the binding protein or a vector containing DNA encoding the binding protein under suitable conditions to produce the binding protein of the invention. See: such as U.S. patent No.4,816,567. Preferred host cells may be CHO cells, NS/0 hybridoma cells, human embryonic kidney 293 cells, E.coli and s.cerevisiae.

With respect to those binding proteins that are antibodies, these antibodies can be produced from genetically engineered animals as fully human antibodies or from phage, yeast, ribosome, or E.coli antibody display libraries. See, for example: clackson et al, Nature 352(1991), 624-628, Marks et al, J.mol.biol.222(1991), 581-597, Feldhaus and SiegeelJ Immunol methods.290, 69-80, Groves and Osbourn, Expert Opin biol. The.5, 125-135and Jostock and Dubel, Comb Chem Highthogh Screen.8, 127-133.

Human antibodies avoid the problems associated with antibodies having murine or rat variable and/or constant regions. The presence of these proteins of murine or rat origin may lead to rapid clearance of these antibodies by the patient or to an immune response against such antibodies. To avoid the use of murine or rat-derived antibodies, fully human antibodies can be generated by introducing a functional human antibody locus into a rodent, other mammal, or animal such that the rodent, other mammal, or animal produces fully human antibodies.

The method of producing the whole human antibody is by usingStrain mice that have been engineered to contain germline-configured fragments of 245kb and 190kb size of the human heavy and kappa-light chain loci. Other XenoMouse strain mice contain 980kb and 800kb sized germline configured fragments of the human heavy chain locus and the kappa-light chain locus. Still other XenoMouse strain mice contain 980kb and 800kb sized germline configuration fragments of the human heavy and kappa-light chain loci plus a 740kb sized germline configuration of the entire human lambda-light chain locus. See Mendez et al, Nature Genetics 15: 146-156(1997) and Green and jakobvits j.exp.med.188: 483-495(1998).Strain mice are available from Abgenix, Inc.

Intensive discussion of mouse productionAnd in the following U.S. patent application serial nos.: 07/466,008, 1995, 12/1990, 07/610,515, 11/8/1990, 07/919,297, 1992, 7/24/1992, 07/922,649, 1992, 7/30/1994, 08/031,801, 1993, 3/15/1993, 08/112,848, 1993, 8/27/1993, 08/234,145, 1994, 4/28, 08/376,279, 1995, 1/20/1995, 08/430,938, 1995, 4/27/464, 584, 1995, 6/5/1995, 08/464,582, 1995, 6/5/486, 582, 08/08, 191, 1995, 6/5/1995, 08/462,837, 1995, 08/462, 853, 486, 855/08, 486, 855, 857, 1995, 857, 1995, 857, 1995, 5, 08/724,752, 6/1995, 2, 10/1996, and 08/759,620, 3, 12/1996; U.S. patent publication nos.: 2003/0217373, filed on 20/11/2002; and U.S. patent nos.6,833,268, 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and japanese patent nos.3068180b2, 3068506B2, and 3068507B 2. See also european patent: EP 0463151B1, grant notice on 12.6.1996; international patent application No., WO 94/02602, published on 3/2 of 1994; international patent application No. WO 96/34096, published in 1996 at 10/31; WO 98/24893, published on 11/6/1998, WO00/76310, published on 21/12/2000. The disclosures of each of the above-referenced patents, patent applications, and documents are hereby incorporated by reference herein in their entirety.

In an alternative approach, other companies, including GenPharm International, have adopted a "mini-locus" approach. In the mini-locus approach, exogenous Ig loci are mimicked by the inclusion of a single gene from an Ig locus. Thus, one or more V_HGene, one or more D_HGene, one or more fruit J_HThe gene, the mu-constant domain, and the second constant domain (preferably the gamma-constant domain) form a construct for insertion into an animal. Such a process is described in the following patents:

U.S. patent 5,545,807 to Surani et al and U.S. patent Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 to Lonberg and Kay; U.S. Pat. Nos.5,591,669 and 6,023.010 to Krimpen and Berns; U.S. Pat. Nos.5,612,205, 5,721,367 and 5,789,215 to Berns et al and U.S. Pat. Nos.5,643,763 to Choi and Dunn; and Genpharm International U.S. patent application Ser. No. 07/574,748, 8/29/1990, 07/575,962, 8/31/1990, 07/810,279, 12/17/1991, 07/853,408, 3/18/1992, 07/904,068, 6/23/1992, 07/990,860, 12/16/1992, 08/053,131, 4/26/1993, 08/096,762, 7/22/1993, 08/155,301, 11/18/1993, 08/161,739, 12/3/1993, 08/165,699, 12/10/1993, 08/209,741, and 3/9/1994, the disclosures of each of which are incorporated herein by reference. See also European patent No.0546073B1, International patent application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884, and U.S. Pat. No.5,981,175; the disclosures of the above references are hereby incorporated by reference in their entirety.

Kirin also describes the production of human antibodies in mice, in which large fragments of chromosomes or whole chromosomes are introduced by minicell fusion. See European patent application Nos.773,288 and 843,961, the disclosures of which are hereby incorporated by reference. Alternative KM^TMMice have also been generated, which is the result of cross breeding of Kirin Tc mice with Medarex mini-locus (Humab) mice. These mice had the transchromosomal HC of Kirin mice and the kappa-light chain of transgenic Metarex mice (Ishida et al, Cloning Stem Cells, (2002) 4: 91-102).

Human antibodies can also be derived by in vitro methods. Suitable examples include (but are not limited to): phage display (commercialized by Cambridge Antibody Technology, Morphosys, Dyax, Biosite/Metarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (commercialized by Cambridge Antibody Technology), yeast display, and the like.

The antibody described in the text, by usingThe technique was prepared as described below. Such mice are capable of producing human immunoglobulin molecules and antibodies, but lack the ability to produce murine immunoglobulin molecules and antibodies. Techniques for achieving the same purpose are disclosed in patents, patent applications, and literature that are disclosed in the background section of the text. However, it is specifically noted. A preferred embodiment for generating transgenic mice and antibodies therefrom is disclosed in U.S. patent application Ser. No.08/759,620, filed on 3.12.1996, and published in International patent application No. WO 98/24893 on 11.6.1998 and International application No. WO00/76310 on 21.12.2000, the disclosures of which are incorporated herein by reference. See also Mendez et al, Nature Genetics 15: 146-156(1997), the disclosure of which is hereby incorporated by reference.

By using this technique, whole human monoclonal antibodies against a range of antigens are generated. In essence, the temperature of the molten steel is controlled,the lineage mice are immunized with an antigen of interest (e.g., HER-3), lymphocytes (e.g., B cells) are recovered from the antibody-expressing mice, and the resulting cell lines are then fused with a myeloid cell line to produce immortalized hybridoma cell lines capable of producing specific antibodies to the antigen of interest. Provided herein are methods for the production of multiple myeloma cell lines that produce antibodies specific for HER-3. In addition, methods for characterizing antibodies produced by such cell lines, including analysis of the nucleotide and amino acid sequences of the heavy and light chains of such antibodies, are also provided herein.

Generally speaking, general speaking, TongThe antibodies produced by the over-fused hybridoma cells were human IgG1 heavy chain and intact human kappa-light chain. The antibodies described herein have a human IgG4 heavy chain and an IgG1 heavy chain. Antibodies may also be of other human isotypes, including IgG2 and IgG 3. These antibodies have high affinity, typically from about 10-⁶To greater than 10^-13M or a KD value below.

Another aspect of the invention relates to an isolated nucleic acid molecule encoding a binding protein of the invention. The term "isolated nucleic acid molecule" as used in the context of the present invention refers to a genomic, cDNA, or synthetic polynucleotide or some combination thereof, which, depending on its source, "isolated nucleic acid molecule" (1) does not bind to all or part of a polynucleotide in which an "isolated polynucleotide" is naturally found, (2) is operably linked to a polynucleotide with which it is not naturally associated, or (3) does not occur as part of a larger sequence in nature. Furthermore, the term "nucleic acid molecule" as used herein refers to a polymeric form of nucleotides of at least 10 bases in length, which may be ribonucleotides or deoxyribonucleotides or modified forms of either nucleic acid type, e.g., nucleic acids containing modified or substituted sugar groups and the like. The term also includes DNA in single-stranded or double-stranded form.

In one embodiment of the invention, the nucleic acid molecule of the invention is operably linked to a control sequence. The term "control sequences" as used herein refers to polynucleotide sequences necessary for expression and manipulation of a linked coding sequence. The nature of such control sequences varies depending on the host organism. In prokaryotes, such control sequences typically include a promoter, ribosome binding site, and transcription termination sequence; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. According to the invention, the term "control sequences" shall at least include all the components necessary for the expression and processing, and may also include other beneficial components, such as: the leader sequence and the fusion partner sequence. Further, the term "operably linked" as used herein refers to the positioning of the elements in a relationship that allows them to function in the intended manner. Furthermore, according to the present invention, an expression control sequence is operably linked to a coding sequence in such a way that the coding sequence is expressed, under conditions compatible with the expression control sequence.

Another aspect of the invention is a vector comprising a nucleic acid molecule encoding a binding protein of the invention. The nucleic acid molecule can be operably linked to a control sequence. In addition, the vector may contain an origin of replication or a selectable marker gene. Examples of carriers which can be used according to the invention are: plasmids, cosmids (cosmids), phages, viruses, and the like.

Another aspect of the invention relates to host cells transformed with the plasmids or nucleic acid molecules of the invention. Transformation can be accomplished by any well-known method for introducing a polynucleotide into a host cell, including, for example: the polynucleotides are packaged in viruses (or in viral vectors) and host cells are transduced with the viruses (or vectors), or by transfection procedures well known in the art, as exemplified by U.S. Pat. Nos.4,399,216, 4,912,040, 4,740,461, and 4,959,455. These patents are incorporated herein by reference. Specifically, methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, packaging of the polynucleotide in liposomes, and direct injection of DNA into the nucleus. Examples of host cells which can be used according to the invention are hybridoma eukaryotic cells such as mammalian cells, for example: hamster, rabbit, rat, pig, mouse, or other mammalian cell; plant cells and fungal cells, such as corn, tobacco, saccharomyces cerevisiae, pichia pastoris; prokaryotic cells such as E.coli; and other cells used in the art for antibody production. In particular, mammalian cell lines useful as expression hosts are well known in the art and include a number of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human liver cancer cells (e.g., HepG2), and a number of other cell lines.

Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient at least one binding protein of the invention together with a pharmaceutically acceptable carrier, diluent and/or adjuvant. The term "pharmaceutical composition" as used herein refers to a compound or component that induces a desired therapeutic effect when properly administered to a patient (The McGraw-Hill Dictionary of chemical Terms, Parker, s., ed., McGraw-Hill, San Francisco (1985), incorporated herein by reference). According to the invention, the efficacy of the pharmaceutical composition of the invention is based on the binding of at least one binding protein to HER-3. Preferably, such binding results in a reduction of HER-3 mediated signal transduction.

In addition, the term "carrier" as used herein includes carriers, pharmaceutical excipients, or stabilizers which do not harm the cells or mammal to which they are exposed at the dosages and concentrations employed. Typically the physiologically acceptable carrier is an aqueous pH buffered solution or liposomes (vesicles composed of various lipids, phospholipids and/or surfactants, useful for drug delivery to mammals). Examples of the physiologically acceptable carrier include buffers such as phosphoric acid, citric acid and organic acids; antioxidants include ascorbic acid; low molecular (less than ten residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, arginine or lysine; monosaccharides, polysaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN (TWEEN)^TM) Polyethylene glycol (PEG) and PLURONICS^TM。

In one embodiment of the invention, at least one binding protein of the invention comprised in the pharmaceutical composition is coupled to an effector group, such as e.g. calicheamicin, Auristatin-PE, a radioisotope or a toxic chemotherapeutic agent such as geldanamycin and maytansine. In particular, conjugates of these binding proteins are useful for their elimination in target cells expressing HER-3 (e.g., cancer cells).

Furthermore, the binding proteins of the invention provide advantages for attaching radioisotopes, for example, for the treatment of tumors. Unlike chemotherapy or other cancer treatments, radioimmunotherapy or administration of a combination of radioisotope binding proteins is directed to cancer cells with minimal damage to surrounding normal healthy tissue. With such "magic bullets," patients can be treated with very small amounts of radioisotopes compared to other treatment modalities available today. Preferred radioisotopes include yttrium 90: (⁹⁰Y), indium¹¹¹(¹¹¹In)、¹³¹I、⁹⁹mTc, radioactive silver-111, radioactive silver-199, and Bismuth²¹³. The linkage of isotopes to the binding proteins of the invention may, for example, be performed using conventional bifunctional chelating agents. Because silver is monovalent, for the attachment of radioactive silver-111 and radioactive silver-199, a sulfur-based linker can be used (Hazra et al, Cell Biophys.24-25, 1-7 (1994)). Linking of the silver isotope may include reduction of immunoglobulins with ascorbic acid. Furthermore, tiuxetan is a MX-DTPA linker chelator that attaches to Ibritumomab to form Ibritumomab tiuxetan (Zevalin) (Witzig, T.E, Cancer Chemother. Pharmacol.48suppl 1, 91-5 (2001). Ibrititumomab tiuxetan can be conjugated with isotopes such as indium¹¹¹(¹¹¹In) or⁹⁰Y is reacted to form¹¹¹In-ibritumomab tiuxetan and⁹⁰Y-ibritumomabtiuxetan。

furthermore, the binding proteins of the invention, particularly when used to treat cancer, may be conjugated with toxic chemotherapeutic drugs such as calicheamicin (Hamann et al, biocononjug. chem.13(1), 40-6(2002), geldanamycin (Mandler et al, J.Natl. cancer Inst, 92(19), 1549-51(2000)) and maytansinoids, such as maytansinoid drug DM1(Liu et al, Proc.Natl.Acad.Sci.U.S.A.93: 8618-8623(1996)), different linkers that release the drug under acidic or reducing conditions or upon exposure to specific proteases.

For example, Auristatin-PE, an antimicrotubule agent, is a structural modification of dolastatin 10, a marine crustacean-free peptide component. Auristatin-PE has anti-tumor activity and anti-tumor vascular activity (Otani et al, jpn. j. cancer res.91(8), 837-44 (2000)). For example, Auristatin-PE inhibits cell growth and induces cell cycle arrest and apoptosis in pancreatic cancer cell lines (Li et al, int.j.mol.med.3(6), 647-53 (1999)). Accordingly, in order to specifically direct the antitumor activity and antitumor vascular activity of auristatin-PE to specific tumors, auristatin-PE may be conjugated to a binding protein of the present invention.

In one embodiment of the invention, the pharmaceutical composition comprises at least one additional active ingredient. Examples of further active ingredients which can be used according to the invention are antibodies or low molecular weight inhibitors of other receptor protein kinases (e.g. EGFR, HER-2, HER-4, IGFR-1), or c-met, receptor ligands such as Vascular Endothelial Growth Factor (VEGF), cytotoxic agents such as doxorubicin, cis-diaminedichloroplatinum or carboplatin, cytokines or antineoplastic drugs. Many antineoplastic agents are currently well known in the art. In one embodiment, the antineoplastic agent is selected from the group of therapeutically effective proteins, including but not limited to antibodies or immunomodulatory proteins. In another embodiment, the antineoplastic agent is selected from the group consisting of small molecule inhibitors or chemotherapeutic agents, including mitotic inhibitors, kinase inhibitors, alkylating agents, antimetabolites, chimeric antibiotics (intercalating antibiotics), growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylation inhibitors, anti-survival agents, biological response modifiers, anti-hormones, e.g., anti-androgens, anti-angiogenic agents. When the anti-tumor agent is radiation, treatment may be effected either endogenously (brachytherapy: BT) or exogenously (external radiation therapy: EBRT).

The pharmaceutical compositions of the invention are particularly useful in the diagnosis, prevention or treatment of proliferative diseases. The proliferative disease may be, for example: associated with enhanced HER family signaling. In particular, such diseases may be associated with an increased degree of phosphorylation of HER-3, an increased degree of complex formation between HER-3 and other members of the HER family or with an increased PI 3-kinase activity and/or an increased c-jun-terminal kinase activity and/or AKT activity and/or an increased ERK2 activity and/or PYK2 activity. Preferably, the proliferative disease is selected from the group consisting of: breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, testicular cancer, soft tissue sarcoma, head and neck cancer, other cancers that express or overexpress HER-3, and tumor metastasis formation.

According to the present invention, the term "prevention or treatment" as used herein refers to both pharmacotherapy and prophylactic or preventative measures, wherein a patient in need thereof prevents or slows down (attenuates) a targeted pathological condition or disorder. Patients in need of prevention or treatment include those already exhibiting dysfunction and those prone to dysfunction or those in which dysfunction may be prevented. A patient in need of prophylaxis or treatment is a mammalian patient, that is, any animal classified as a mammal, including humans, domestic and farm animals, zoo animals, racing animals, and pets, such as dogs, cats, sheep, horses, sheep, pigs, goats, rabbits, and the like. Preferably, the animal in need thereof is a human patient.

According to the present invention, the pharmaceutical compositions of the present invention may be formulated by mixing the active agent with physiologically acceptable carriers, diluents and/or adjuvants, and optionally other ingredients commonly included in formulations for providing improved transfer, release, tolerance, and the like. The pharmaceutical compositions of the invention may be configured, for example, as: in the form of frozen formulations, aqueous solutions, dispersions or solid preparations, such as tablets, dragees or capsules. Many suitable formulations can be found in formulary sets well known to all pharmacy: remington's Pharmaceutical sciencesces (18 th edition, MackPublishing Company, Easton, Pa. (1990)), particularly chapter 87 written therein by Block, Lawrence. These formulations include, for example: powders, pastes, ointments, gels, waxes, oils, lipids, and (cationic or anionic) lipids containing particles (e.g. Lipofectin)^TM) DNA conjugates, anhydrous sorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbonized waxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbonized waxes. Any of the foregoing mixtures are suitable in the treatment and therapy according to the present invention, provided that the active ingredients in the formulation are not inactivated by the formulation, and the manner of administration of the formulation is physiologically compatible and tolerable. See also Baldrick p., "pharmaceutical lexcipient definition: the new for clinical guide. ", Regul. Toxicol. Pharmacol.32(2), 210-218 (2000); wang W., "lysine and concentration of soluble proteinaceous pharmaceuticals.", int.J.pharm.203(1-2), 1-60 (2000); charman w.n., "Lipids, lipid-fatty drives, and oral-fatty-recording contacts,", j.pharm.sci.89(8), 967-978 (2000); powell et al, "Compendium of excipients for formulations", PDA J.pharm.Sci.Technol.52, 238-311 (1998); additional information concerning formulations, adjuvants and carriers cited in this text is well known to the medicinal chemist.

Another aspect of the present invention relates to the use of at least one isolated binding protein of the invention and optionally at least one further active ingredient, e.g. at least one antineoplastic drug as described above, in admixture with a pharmaceutically acceptable carrier, diluent and/or adjuvant, for the manufacture of a pharmaceutical composition species for the diagnosis, prevention and treatment of hyperproliferative diseases. Optionally, the pharmaceutical composition is a pharmaceutical composition as described above, and the hyperproliferative disease is one of the hyperproliferative diseases mentioned above.

Another aspect of the invention relates to a method for diagnosing a disease or a condition associated with HER-3 expression comprising contacting a sample with a sample comprising a binding protein of the invention and detecting the presence of HER-3 in the sample. The sample may be a HER-3 expressing cell, such as a tumor cell, a blood sample, or other suitable sample. In a preferred embodiment of the invention the disease or condition associated with HER-3 expression is a hyperproliferative disease as defined above.

According to the invention, such a method can, for example: for the detection of HER-3 antigen in cells, for the determination of the concentration of HER-3 antigen in a patient suffering from the hyperproliferative disease mentioned above or for the staging of the hyperproliferative disease in a patient. To stage the development of a proliferative disease in a subject of interest, or to define the response of a subject of study to a treatment stage, a sample of blood may, for example, be taken from the subject, the concentration of HER-3 antigen present in the sample also being determined. The resulting concentrations were used to identify in which range of concentrations the values dropped. The identified range correlates with the stage of development or with the stage of treatment identified in various populations of diagnosed subjects, thus providing a stage in the study subject. Biopsy of diseases (e.g., cancer), tissues from patients can also be used to assess the amount of HER-3 antigen present. The amount of HER-3 antigen present in diseased tissue can be assessed by immunohistochemistry, ELISA, or antibody analysis using the HER-3 antibodies of the invention. Other parameters of diagnostic interest are the dimer status and the ligand of the dimer of the HER-3 protein and the activation status of it and its ligands. Methods for protein analysis to determine these parameters are well known in the art, among which are western blot and co-immunoprecipitation techniques, FACS analysis, chemical cross-linking, Bioluminescence Resonance Energy Transfer (BRET), Fluorescence Resonance Energy Transfer (FRET), and the like (e.g., Price et al, Methods in Molecular Biology, 218: 255-268(2002) or the eTag technology (WO 0503707, WO04091384, WO 04011900).

Furthermore, another aspect of the invention relates to a method for preventing or treating a disease or condition associated with HER-3 expression in a patient, comprising administering to a patient in need thereof an effective amount of at least one binding protein of the invention. Preferably, the disease or condition associated with HER-3 expression is a hyperproliferative disease as defined above. The subject in need of prophylaxis or treatment according to the present invention is a mammalian subject, i.e. any animal classified as a mammal, including humans, domestic and farm animals, zoo animals, sports animals and pets, such as dogs, cats, sheep, horses, sheep, pigs, goats, rabbits and the like. Preferably, the animal in need thereof is a human patient.

In a preferred embodiment of the present invention, a method for preventing or treating a hyperproliferative disease in a patient in need thereof comprises: an effective amount of at least one binding protein of the invention is administered to the patient, plus at least one other active ingredient, such as at least one of the above-mentioned antineoplastic agents. Preferably, the method is used to inhibit abnormal cell growth, migration or invasion.

In addition to the classical mode of administration of potential binding protein therapies, for example by the modes mentioned above, newly developed forms of administration are also useful according to the present invention. For example, local administration of 131I-labeled monoclonal antibodies for the treatment of primary brain tumors after surgical resection has been reported. In addition, direct stereotactic intracerebral injection of monoclonal antibodies and fragments thereof has also been studied clinically or preclinically. The hypertonic perfusion of carotid arteries with drugs conjugated with human monoclonal antibodies is an experimental strategy for primary brain malignancies.

Depending on the type of patient being treated and the severity of the condition, about 1. mu.g/kg to 15mg/kg of at least one binding protein of the invention may be administered to a patient in need thereof, for example: by one or more separate administrations or continuous infusion. Typical daily dosages will be in the range of about 1. mu.g/kg to 100mg/kg or higher, depending on the factors mentioned above. Repeated administrations over several days or longer, depending on the condition of the patient being treated, continue until the desired reduction in disease symptoms occurs.

The dosage of the at least one antineoplastic agent administered depends on a number of factors. For example, the nature of the drug, the type of tumor, or the route of administration. It must be emphasized that the present invention is not limited to any dosage.

Finally, another aspect of the invention relates to a kit for the diagnosis, prevention and treatment of hyperproliferative diseases associated with HER-3 mediated signal transduction comprising at least one binding protein and/or nucleic acid molecule and/or vector of the invention. In addition, the kit of the invention may further comprise at least one other active agent, such as: at least one of the other antineoplastic agents mentioned above.

The invention will be further illustrated by the following examples and the accompanying figures.

The following examples, including experiments conducted and results obtained, are for illustrative purposes only and are not to be construed as limiting the invention.

CDNA for Domain (ECD) obtained by Polymerase Chain Reaction (PCR) from pcDNA3-HER-3 (expression vector containing full-length human HER-3, C.Walasch et al, EMBO J.14, 4267-4275) using primers based on the HER-3 sequence (Genebank accession number NM-001982).

The primers used for amplification of HER-3 were as follows:

a forward primer: 5' -CGGGATCCATGTCCTAGCCTAGGGGC-3′(SEQ ID NO：233)

Reverse primer: 5' -GCTCTAGATTAATGATGATGATGATGATGTTGTCCTAAACAGTCTTG-3′(SEQ ID NO：234)

The PCR product was digested with BamH1 and XbaI, then ligated into BamH1 and XbaI digested pcDNA3 (Invitrogen). Plasmids were transfected into HEK293 cells by the calcium phosphate method. HER-3-HIS fusion protein was purified from the culture medium of the harvest conditions by Ni-NTA affinity chromatography.

Rat 1HER-3 cells were obtained by retroviral gene transfer. Briefly, GP + E86 cells (3X 10)⁵One) were inoculated in a 60 mm culture dish and then transfected by calcium phosphate method with 2. mu.g/ml pIXSN vector or pIXSN-HER-3cDNA (C.Wallacch, PhD Thesis, Max-Planck institute of Biochemistry, Martinsried, Germany). After 24 hours, the medium was replaced with fresh medium and GP + E86 cells were cultured for 4 to 8 hours. In thatNear confluent Rat1 cells (2X 10 per 6 cm dish) in the presence of coagulated polyamine (4 mg/ml; Aldrich)⁵Individual cells) and then incubated with supernatants of GP + E86 cells releasing high titers of pLXSN or pLXSN-HER-3, picornavirus C for 4 to 12 hours. After medium exchange, selection of Rat1 cells with G418 was initiated. Typically, stable clones were picked after 21 days.

Example 2: expression of HER-3 in human cancer cell lines.

Receptor tyrosine kinases, such as HER-3, play a crucial role in the initiation and progression of hyperproliferative diseases, such as the transition from benign proliferative cell growth to malignancy. Because the expression of HER-3 differs in tumor cells and normal cells, analysis of HER-3 expression is an important factor in identifying patient subpopulations that would benefit from treatment with a binding protein of the invention. Therefore, the quantitative expression of HER-3 in a group of cancer cell lines can illustrate the role of HER-3 in human carcinogenesis. Cancer cell lines were cultured under the conditions recommended by ATCC. In detail, 10⁵Individual cells were collected with 10mM EDTA in PBS, washed once with FACS buffer (PBS, 3% FCS, 0.4% azide), and then seeded in round bottom 96-well plates. The cells were centrifuged at 1000rpm for 3 minutes to remove the supernatant and then resuspended in the alpha-HER-3 antibody 2D1D12(WO 03013602) (3. mu.g/ml). The cell suspension was incubated on ice for 1 hour, washed twice with FACS buffer, and then resuspended in donkey anti-human-PE antibody (100. mu.l/well) diluted 1: 50 in FACS buffer. The cell suspension was incubated on ice and in the dark for 30 minutes, washed twice with FACS buffer and then analyzed (FACS, Beckman Coulter). FIG. 1 shows representative results of the analysis, indicating that HER-3 is expressed in a range of human cancers.

Example 3: immunization and potency assay

HER-3E as described in example 1CD protein and C32 cells (human melanoma, ATCC # CRL-1585) were prepared for use as antigens. anti-HER-3 monoclonal antibodies by sequential immunizationMouse (A)Strain: XMG1 and XMG4, Abgenix, inc. fremont, CA).Animals were immunized by injection via the plantar route. The total amount per injection was 50. mu.l per mouse and 25. mu.l per footpad.

For group #1(10XMG1 mice), each mouse was initially immunized with 10 μ g of the HER-3ECD protein and TITERMAX(Sigma, Oakville, ON) in a 1: 1 volume ratio. The next 5 boosts were performed with a mixture of 10. mu.g of HER-3ECD protein and 100. mu.g of alum gel (Sigma, Oakville, ON) (volume ratio 1: 1). The 6 th booster immunization consisted of 10. mu.g of HER-3ECD protein and TITERMAXAccording to the volume ratio of 1: 1. The 7 th injection consisted of 10 μ g of HER-3ECD protein to 100 μ g of alum gel 1: 1 (by volume). The final boost was performed with 10. mu.g of HER-3ECD protein in pyrogen-free DPBS without immunoadjuvant. According to the procedure, the process is carried out,mice were immunized on days 0,4, 7, 11, 15, 20, 24, and 29 and fused on day 33. Two bleeds were through a retrobulbar bleed procedure after the fourth boost on day 13 and after the sixth boost on day 19. There is no group # 2.

For group #3(10XMG1 mouse) and group #4(10XMG4 mouse), each mouse was given a first injectionIrradiation was with 10 in Dulbecco's PBS (DPBS) without a heat source⁷C32 cells and TITERMAXMixing according to the volume ratio of 1: 1. Four subsequent boosts were performed on each mouse with 10 of the non-pyrogenic DPBS⁷A mixture of C32 cells with 25. mu.g of Adju-Phos and 10. mu.g of CpG. Sixth boost immunization each mouse was with 10 of non-pyrogenic DPBS⁷C32 cells and TITERMAXMixing according to the volume ratio of 1: 1. Seventh, eighth, and ninth boosts were performed with 10 of non-pyrogenic DPBS per mouse⁷A mixture of C32 cells with 25. mu.g of Adju-Phos and 10. mu.g of CpG. The tenth to fourteenth boosts were performed on each mouse with 5. mu.g of HER-3ECD protein in DPBS without a pyrogen in combination with 25. mu.g of Adju-Phos and 10. mu.g of CpG. The last boost consisted of 5 μ g of HER-3ECD protein in DPBS without a pyrogen, and no immune adjuvant. Of groups # 3and #4Mice were immunized on days 0, 3,7, 11, 14, 17, 21, 24, 28, 33, 35, 38, 42 and 45 according to this procedure and fusion was performed on day 49. Three bleeds were through a retrobulbar bleed procedure after the fourth boost on day 12 and after the sixth boost on day 19 and after the 12 th boost on day 40.

Selection of animals for antibody harvesting by Titer。

For group 1, anti-HER-3 antibodies are immunizingThe titers in the mouse sera were determined by an anti-HER-3 ECD protein ELISA assay. Each kind ofThe specific titer of the animals was determined by optical density at 650nm,the results are shown in Table 1 below. The titer value is the reciprocal of the maximum dilution of serum with an OD reading of two times background. Thus, the greater the number, the greater the humoral immunity to HER-3 ECD.

TABLE 1 group #1, XMG1

For groups # 3and #4, the anti-HER-3 antibody is immunizingTiters in mouse sera were determined by FACS using Rat1/HER-3 cells (antigen positive cell line) and Rat1/pLSXN cells (antigen negative cell line). Data are presented as geometric mean (GeoMean) fluorescence intensity of anti-HER-3 cells stained with a series of diluted serum samples.

TABLE 2

Group #3.XMG1

TABLE 3

Group #4.XMG4

Example 4: recovery of lymphocytes, B cell isolation, fusion and hybridoma cell production.

Immunized mice were sacrificed and lymph nodes from each group were collected and pooled. Lymphocytes were dissociated by abrasion in DMEM and released from the tissue, which were suspended in DMEM. The cells were counted, and 0.9ml of DMEM per 1 million lymphocytes was added to the cell pellet, and the cells were gently and sufficiently resuspended. Using 100. mu.l of CD90+ magnetic beads per 1 million cells, the cells were labeled by incubation with the magnetic beads for 15 minutes at 4 ℃. Contains up to 10⁸One positive cell (or the total number reaches 2X 10)⁹Individual cells) was loaded onto LS + columns and the columns were rinsed with DMEM. All effluents were collected as CD90 negative fractions (most of these cells were expected to be B cells).

The fusion procedure was performed by mixing 1: 1 the washed enriched B cells above with non-secreted myeloma P3X63Ag8.653 cells (purchased from ATCC under accession number CRL 1580) (Kearney et al, J.Immunol.123, 1979, 1548-1550). The cell mixture was gently pelleted by centrifugation at 800 g. After complete removal of the supernatant, the cells were treated with 2 to 4ml of pronase solution (Calbiochem, Cat. No. 53702, 0.5mg/ml in PBS) for 2 minutes. Then, 3 to 5ml of FBS was added to stop the enzyme activity, and the supernatant was adjusted to a total volume of 40ml with an electric cell fusion solution, ECFS (0.3M sucrose, Sigma, Cat. No. S7903, 0.1mM magnesium acetate, Sigma, Cat. No. M2545, 0.1mM calcium acetate, Sigma, Cat. No. C4705). The supernatant was removed by centrifugation and the cells were then resuspended in 40ml ECFS. This washing step was repeated once, and then the cells were resuspended in ECFS to a concentration of 2X 10⁶Individual cells/ml.

The electrofusion of cells was performed by a fusion generator (model ECM2001, Genetronic, inc., San Diego, CA). The fusion chamber size used was 2.0ml, set by the following instrument: adjusting conditions: voltage: 50 volts, time: 50 seconds; cell membrane disruption: voltage: 3000 volts for 30 microseconds; duration after fusion: for 3 seconds.

After electrofusion, the cell suspension was carefully removed under sterile conditions and then transferred to a sterile tube containing the same volume of hybridoma culture medium (DMEM (JRHBOSCES), 15% FBS (Hyclone), supplemented with L-glutamine, pen/strep, OPI (oxaloacetate, pyruvate, bovine insulin) (both from Sigma) and interleukin-6 (Boehringer Mannheim). The cells were cultured at 37 ℃ for 15 to 30 minutes, then centrifuged at 400g for 5 minutes, the cells were gently resuspended in a small volume of hybridoma selection medium (hybridoma culture medium supplemented with 0.5 XHA (Sigma, Cat. No. A9666), for a total of 5X 10 based on the final total of each 96 well plate⁶The volume of each B cell and 200. mu.l of each well was appropriately adjusted with more hybridoma selection medium. These cells were gently mixed and then added to a 96-well plate to grow. Around 7 or 10 days, half of the medium was removed and cells continued to be fed with hybridoma selection medium.

Example 5: screening of candidate antibodies by ELISA

After 14 days of culture, HER-3 specific antibodies in the supernatant of hybridoma cells from cohort #1 (mice in cohort 1 were randomly divided into fusion #1 and #2) were primary screened by ELISA using purified his-tagged HER-3ECD and back screened against irrelevant his-tagged proteins by ELISA using goat anti-hulgfc-HRP (Caltag inc., catalog No. H10507, diluted 1: 2000 in concentration) to detect binding of human IgG to HER-3ECD immobilized on ELISA plates. According to the preliminary screening results, the old medium supernatant from which the positive hybridoma cells grew was removed, and HER-3 positive hybridoma cells were suspended with fresh hybridoma medium and then transferred to 24-well plates. After two days in culture, these supernatants were ready for a second confirmation screen. In a second determination, HER-3 specific whole human IgGk antibodies were screened, the first positive cells were screened by ELISA, using two sets of detection antibodies: goat anti-hulgGFc-HRP (Caltag Inc., Cat H10507, diluted 1: 2000 using concentration) was used to detect human gamma-chain and goat anti-hlg kappa-HRP (southern Biotechnology, Cat 2060-05) was used to detect human kappa light chain. A total of 91 human IgG/kappa HER-3 specific monoclonal antibodies were generated from cohort # 1.

Example 6: selection of candidate antibodies with FMAT/FACE

After 14 days of culture, the supernatants of cohort # 3and #4 (fusion # 3and #4) hybridomas were screened for HER-3 specific monoclonal antibodies by FMAT. In the first round of screening, hybridoma supernatants were incubated at final 1: 10 dilution with Rat1-Her3 cells expressing human HER-3 and 400ng/ml Cy 5-coupled goat F (ab') 2 anti-human IgG, Fc specific antibody (Jackson ImmunoResearch, Cat. No. 109-176-098) for 6 hours at room temperature. Binding of the antibody and the detection antibody complex to the cells was measured by FMAT (Applied Biosystems). Non-specific binding of the antibody to the cells can be detected by its binding to parental Rat1 cells. A total of 420 hybridomas producing HER-3 specific antibodies were selected from the first round of selection for fusion #3. Supernatants from these expanded cultures were examined by the same FMAT method, with 262 of these being shown to specifically bind HER-3 expressing cells. A total of 193 hybridomas producing HER-3 specific antibodies were selected in the first round of selection for fusion #4. Supernatants from these expanded cultures were assayed by FACS, of which 138 strains were shown to bind specifically to HER-3 expressing cells. In FACS confirmation analysis, Rat1-Xher3 cells and parental Rat1 cells (used as negative controls) were incubated with hybridoma supernatant (diluted 1: 2) in PBS containing 2% FBS at 40 ℃ for 1 hour. After washing with PBS, the binding of antibody to cells was detected with 2.5. mu.g/ml Cy 5-conjugated goat F (ab ') 2 anti-human IgG, Fc-specific antibody (JIR #109-176-098) and 5. mu.g/ml PE-conjugated goat F (ab') 2 anti-human kappa-specific antibody (SB # 2063-09). Unbound antibody was removed by washing with PBS, cells were fixed with 1: 4 diluted cytofix (BD #51-2090KZ) and analyzed with FACS Calibur.

Example 7: by usingSelection of cloned hybridomas

Selecting antibodies derived from groups 1 and 2 for hybridoma cloning based on HER-3 specificity for HER-1(EGFR), HER-2 and HER-4 in ELISA using purified recombinant extracellular protein domains (as available from R & D Biosystems); FACS analysis of human tumor cell lines expressing different HER family members; HER-3 positive cells show a 5-fold increase in mean fluorescence intensity over background in FACS staining. Based on these criteria, a total of 23 hybridomas were selected for cloning using limiting dilution cell plating.

Antibodies derived from groups 3and 4 were used to select hybridoma clones based on HER-3 specificity for HER-1(EGFR), HER-2 and HER-4 and 3 additional criteria. The first criterion was the screening of the antibodies by ELISA using an epitope containing the HER-3L2 domain (see example "structural analysis of anti-HER-3 antibodies" of the invention).

The second criterion is the neutralization of biotin-labeled heregulin-alpha binding to HER-3 expressing cells in FACS-based assays. SKBR-3 cells were collected, washed with medium, pelleted by centrifugation and suspended in medium. Resuspended cells were aliquoted into 96-well cell plates. The cell plate was centrifuged to pellet the cells. The test antibody in the poured hybridoma supernatant was added in an amount of 25. mu.l per well, and the mixture was incubated on ice for 1 hour to allow the antibody to bind. Mu.l of 10nM of a 10nM solution of heregulin-alpha (R & D Biosystems, Minneapolis, MN) was added to each well to a final concentration of 5nM, and then placed on ice for 1.5 hours. The cells were washed with 150. mu.l PBS, pelleted by centrifugation, and the supernatant removed. The cells were resuspended in 50. mu.l of goat anti-HRG-alpha polyclonal antibody at a concentration of 10. mu.g/ml and incubated on ice for 45 minutes. The cells were washed with 200. mu.l PBS, pelleted by centrifugation, and the supernatant was removed. Mu.l of Cy 5-labeled rabbit anti-goat polyclonal antibody (final concentration of 5. mu.g/ml) and 7AAD (final concentration of 10. mu.g/ml) were added and incubated on ice for 15 minutes. The cells were washed with 200. mu.l PBS, pelleted by centrifugation, and the supernatant was removed. Cells were resuspended in 100. mu.l FACS buffer and read by FACS. The HER-3 antibodies tested for reduced binding to heregulin-alpha were those with the lowest fluorescence intensity. A gradient of 10,000 ng/ml to 16ng/ml of mouse HER-3 mab (105.5) or human IgG1HER-3 mab U1-49 diluted 1: 5 was used as a positive control. Negative controls were heregulin-alpha alone, cells alone, goat anti-heregulin-alpha polyclonal antibody alone, and Cy 5-labeled rabbit anti-goat polyclonal antibody alone.

The third criterion is: FACS with cell lines expressing HER-3, relative ordering of affinities and/or higher relative mean fluorescence intensity. The operation of affinity relative ranking was to normalize the concentration of HER-3 specific antibodies and to plot the data obtained from the ELISA below for the restricted antigen.

Normalizing antigen-specific antibody concentrations by high antigen ELISA

The antigen-specific antibody concentration in the supernatant was normalized using an ELISA method. Parallel titer experiments were performed using known concentrations of two anti-HER-3 human IgG1 antibodies in group 1 to generate a standard curve. The amount of antigen-specific antibody in the hybridoma supernatants determined in groups 3and 4 can be compared to a standard curve. Following this method, the concentration of human HER 3IgG antibody in each group of hybridoma cultures was estimated.

Neutral avidin plates were prepared by coating 1 XPBS/8. mu.g/ml neutral antibiotic protein (50. mu.l/well) in 0.05% sodium azide on Costar3368 medium conjugate plates and incubating overnight at 4 ℃. The next day, the plates were blocked with 1 XPBS/1% skim milk. A500 ng/ml photobiotin his-tag-HER-3 ECD in 1 XPBS/1% skim milk was bound to the neutravidin plates by incubation for 1 hour at room temperature. Hybridoma supernatants were diluted 1: 2.5 with 1 XPBS/1% skim milk/0.05% sodium azide, added at 50. mu.l/well from the first 1: 31 to the last 1: 7568, and then allowed to react at room temperature for 20 hours. Gradient dilutions were used to ensure that the OD readings obtained for each unknown sample were within the linear range of the assay. Next, a test secondary goat anti-human IgG in 1 XPBS/1% skim milk at 400ng/ml, Fc HRP was added at 50. mu.l/well. After 1 hour incubation at room temperature, the plates were washed 5 times with water and 50 μ l of a single component TMB substrate was added to each well. After 30 minutes, the reaction was stopped by adding 50. mu.l of 1M hydrochloric acid to each well, and the cell plates were read at a wavelength of 450 nm. A standard curve was generated from two IgG1HER-3 mabs in cohort 1, which were diluted 1: 2 in a gradient from 1000ng/ml to 0.06ng/ml and then estimated by ELISA using the method above. For each unknown sample, the OD readings in the linear range during the analysis were used to estimate the concentration of human HER-3IgG in each set of samples.

Limited antigen analysis is a method of aligning the affinity of antigen-specific antibodies prepared from B cell culture supernatants relative to all other antigen-specific antibodies. When there is very low antigen coating, only the highest affinity antibody is able to bind to any detectable level at equilibrium. (see, for example, PCT publication WO/03048730A2 entitled "IDENTIFICATION OF HIGH AFFINITY Moleculas BY LIMITED dilution screening NG", published 6/12/2003). In this case, the known concentrations and known KD of mabs derived from group 1 were used as a benchmark in the analysis.

Neutral avidin plates were prepared by coating 8. mu.g/ml neutral avidin (50. mu.l/well) in 1 XPBS/0.05% sodium azide in Costar3368 medium conjugate plates and then incubated overnight at 4 ℃. The next day, the plates were blocked with 1 XPBS/1% skim milk. Biotin-labeled his-tagged-HER-3ECD (50. mu.l/well) was bound to 96-well neutravidin plates at 5 concentrations (125, 62.5, 31.2, 15.6, and 7.8ng/ml) in 1 XPBS/1% skim milk for 1 hour at room temperature. Each plate was washed 5 times with water. Hybridoma supernatants diluted 1: 31 with 1 XPBS/1% skim milk/0.05% sodium azide were added at 50. mu.l/well. After incubation for 20 hours at room temperature on a shaker, the plates were washed 5 times with distilled water. Next, a 400ng/ml secondary detection antibody, goat anti-human IgG Fc HRP (horseradish peroxidase), in 1 XPBS/1% skim milk was added at 50. mu.l/well. After 1 hour at room temperature, the plates were washed 5 times with distilled water and 50. mu.l of a one-component TMB substrate was added to each well. After 30 minutes, the reaction was stopped by adding 50. mu.l of 1M hydrochloric acid to each well, and the cell plates were read at a wavelength of 450 nm. OD values in the linear range resulting from OD readings obtained from antigen concentration were used for data analysis.

High antigen data, which allows relative estimates of specific antibody concentrations (see above for details), are plotted against limited antigen OD to describe relatively high affinity antibodies, e.g., those that bind have a higher OD in limited antigen assays when the supernatant has a lower amount of IgG HER-3 antibody.

In these assays, the 33 best expressing antibodies from groups 3and 4 were further cloned by limiting dilution hybridoma plating.

Alternatively, FACS analysis of HER-3 expression in Rat1/pLXSN and Rat1/HER-3 cells showed similar results (no cross-reactivity with endogenous Rat epitopes) (FIG. 2).

In detail, 1X 10 EDTA in PBS was collected⁵Cells were washed once with FACS buffer (PBS, 3% FCS, 0.4% azide) and then seeded in 96-well round bottom plates. These cells were centrifuged at 1000rpm for 3 minutes to remove the supernatant and then resuspended in specific HER-family antibody (3. mu.g/ml). The cell suspension was incubated on ice for 45 minutes, washed twice with FACS buffer, and then resuspended in cells using a 1: 50 dilution of secondary antibody (100. mu.l/well) donkey anti-human-PE (Jackson Immunoresearch, Pa.). These cell suspensions were incubated on ice and in the dark for 30 minutes, washed twice with FACS buffer and analyzed (FACS, Beckman Coulter).

Example 8: structural analysis of anti-HER-3 antibody of the invention

The following discussion provides information regarding the structure of antibodies produced by the present invention. To analyze the structure of the antibodies produced by the present invention, genes encoding the heavy and light chain fragments were amplified from specific hybridomas. Sequencing was accomplished as follows:

hybridization from 96-well plates individually by reverse transcriptase polymerase chain reaction (RT-PCR)The tumor clones amplified VH and VL transcripts. From about 2X 10 with Fast-Track kit (Invitrogen)⁵Poly (A) + -mRNA was isolated from each hybridoma cell. 4 sets of PCR reactions were run for each set of hybridoma cells: two groups were used for the light chain (κ) and two groups for the heavy chain (γ). OneStep room temperature PCR kit from QIAGEN was used for amplification (QIAGEN, catalog No. 210212). In this coupled room temperature PCR reaction, cDNA was synthesized by a mixture of room temperature enzymes (Omniscript and Sensticript) using specific primers corresponding to C-. kappa.or to the antisense sequence of the consensus sequence of the CH1 region of the C.gamma.gene. The reverse transcription was completed in 1 hour at 50 ℃ and then the cDNA was amplified by PCR using a highly specific and highly sensitive hot-start Taq DNA polymerase. A mixed 5' -sense primer was used for each PCR reaction, the sequence of which was based on the leader sequence of VH and VK in the Vbase site (http:// Vbase. mrc-cpe. cam. a. c.uk /).

The PCR reaction was run first with a hot start at 94 ℃ for 15 minutes, followed by 40 cycles: 94 ℃,30 seconds (denaturation), 60 ℃,30 seconds (annealing) and 72 ℃,1 minute (extension).

The PCR products were purified and then directly sequenced by ABI PRISMBIGDY terminator cycle sequencing reaction kit (Perkinelmer) using forward and reverse PCR primers. Both strands were sequenced using Prism dye-terminator sequencing kit and ABI 377 sequencer.

Sequence analysis

Analysis of the human V heavy chain and vk cDNA sequences of HER3 antibodies was done by aligning the HER-3 sequences with human germline V heavy chain and vk sequences using the self-developed software Abgnix5 AS. The alignment software identified the use of V, D and J genes, as well as nucleic acid insertions at the recombination junctions and somatic mutations. The computer generated amino acid sequences were also used to identify somatic mutations. Using commercially available sequence analysis software and publicly available information of human V, D and J genes, for example: vbase (http:// Vbase. mrc-cpe. cam. ac. uk /), similar results were obtained.

Molecular cloning of monoclonal antibody U1-59

Total RNA was extracted from tissue culture wells containing multiple hybridoma lineages, including the hybridoma lineage that secretes antibody U1-59. The heavy chain variable region was amplified with 5 '-leader VH family primer and 3' -C-gamma primer. One major band was amplified with the VH4 primer, and no other bands were seen. The VH4-34 γ fragment was cloned in-frame with the human γ 1 constant region gene of the pCDNA expression vector.

IgM heavy chain variable region was amplified with 5 'VH family specific primers and 3' mu constant region primers. One major band was amplified with the VH2 primer, and no other bands were seen. The VH2-5mu fragment was cloned in-frame into the human mu constant region gene of the pCDNA expression vector. Amplification and sequencing of the V.kappa.chain. Four kappa chain RT-PCR products were identified. The product was sequenced and the in silico translated sequences were analyzed, 3 of which contained open reading frames. These 3 functional kappa chains were cloned into oligo clone U1-59 hybridoma wells identified based on the V kappa genes as (1) VK1A3-JK2, (2) VK1A20-JK 3and (3) B3-JK 1. All vks were cloned in-frame to the human kappa light chain constant region gene of the pCDNA expression vector.

Transfection

Each heavy chain and each kappa light chain were transfected, resulting in a total of 6 heavy/kappa light chain pairs in transient transfection. Transfection of both the gamma chain and the a20 kappa chain resulted in poor antibody expression, with no antibody secretion or no antibody detection when the a20 kappa chain was co-transformed with the mu chain. A total of 3IgG subpopulations and two IgM subpopulations were suitable for HER-3 binding assays.

Chain	VH	D	J	Constant region	ORF
						Heavy chain	VH4-34	D1-20	JH2	γ	Is that
Heavy chain	VH2-5	D6-6	JH4b	Mu	Is that
						Light chain	A3		JK2	κ	Is that
Light chain	A20		JK3	κ	Is that
						Light chain	B3		JK1	κ	Is that
Light chain	A27		JK3	κ	Whether or not

Binding activity to HER-3 positive cell lines was detected by FACS using IgG1 monoclonal antibody consisting of VH4-34 and B3 kappa chains. No other VH/V κ combinations produced fluorescence signals above background when FACS was performed with HER-3 positive cell lines.

Binding competition for anti-HER-3 antibodies

Clusters of HER-3 antibodies that compete for binding to HER-3 were evaluated using multiple competitive antibody binding in J immunological methods.288, 91-98(2004) published by Jia et al. The experimental HER-3 antibodies derived from group 1 aggregated into 5bins based on binding competition.

Bin#1	Bin#2	Bin#3	Bin#4	Bin#5
					U1-42	U1-48	U1-52	U1-38	U1-45
U1-44	U1-50		U1-39	U1-40
					U1-62	U1-51			U1-41
U1-46				U1-43
					U1-47	U1-49			U1-61
U1-58				U1-53
									U1-55

Epitope identification of anti-HER-3 antibodies

Epitopes of the human anti-HER-3 antibody of the invention have been identified. First, reduced and denatured HER-3-His tag-purified ECD proteins were analyzed by point hybridization with tested anti-HER-3 antibodies (U1-59, U1-61, U1-41, U1-46, U1-53, U1-43, U1-44, U1-47, U1-52, U1-40, U1-49), demonstrating that all epitopes are sensitive to reduction of disulfide bonds, indicating that they all have discrete epitopes. Next, antibody-defining domains are located on the HER-3 molecule by various engineered human-murine HER-3 chimeric antibodies, which are divided into four domains based on the HER-3 extracellular domain:

1) l1 (D1): a secondary ligand-binding domain which is,

2) s1 (D2): the first cysteine-rich domain of the protein,

3) l2 (D3): a major ligand domain, and

4) s2 (D4): a second cysteine-rich domain.

The cDNA for the extracellular domain (ECD) of human HER-3 was amplified from RAT1-HER-3 cells. Rat HER-3cDNA was amplified from murine liver RNA by RT-PCR and then determined by sequencing. The cDNAs expressing human ECD and rat Her3 were cloned into mammalian expression vectors to form V5-His fusion proteins. The domain from human HER-3ECD was replaced into the scaffold protein provided by rat HER-3ECD by using Mfe1, BstX1 and DraIII endogenous cleavage sites. In this way, various chimeric rat/human HER-3ECD HIS fusion proteins (amino acids 1-160, 161-358, 359-575, 1-358, 359-604) were constructed and expressed by transiently infecting HEK293T cells. The expression of the constructed protein was determined using a rat monoclonal antibody against human HER-3. The binding of human monoclonal antibodies to secreted chimeric ECDs was tested by ELISA.

Two human antibodies, including antibody U1-59, cross-react with rat HER-3. To specify the binding domain, the antibody activity of these monoclonal antibodies was determined against a truncated form of HER-3, wherein the HER-3 truncated form consists of the protein tagged with L1-S1-V5 his, purified from the supernatant of HEK293T cells transfected with plasmid DNA encoding the extracellular domain of HER3L 1-S1. In ELISA experiments, the monoclonal antibody U1-59 binds to L1-S1 protein, and the epitope is shown to be L1-S1. mab2.5.1 did not bind to L1-S1 protein, indicating that its epitope is located at L2-S2. Further localization of antibody U1-59 was accomplished by on-chip protease digestion using SELDI time-of-flight mass spectrometry and the monoclonal antibody-HER-3 ECD complex.

Localization of U1-59 epitopes Using SELDI

The U1-59 antibody was further mapped by on-chip protease digestion using SELDI time-of-flight mass spectrometry and the monoclonal antibody-HER-3 ECD complex. Protein A is covalently bound to the PS20 protein chip array for capturing monoclonal antibody U1-59. The PS20 protein chip complex with monoclonal antibody was then incubated with HER-3-His purified protein. The antibody-antigen complex is then digested with a high concentration of Asp-N. The chip was washed, so that only HER-3 remained on the chip. Epitopes were determined by SELDI and fragments were identified by mass spectrometry. The 6814D fragment identified corresponded to the two potentially desirable polypeptides produced by partial digestion of the HER-3-his ECD. Both overlapping polypeptides are located with the S1 domain. The epitope is positioned from position 251 to 325 by combining SELDI and the results with the constructed HER-3 deletion.

The positions of the HER-3 extracellular part-binding domains recognized by the human anti-HER-3 monoclonal antibodies of the invention are summarized in Table 4. The localization of the epitope domain is consistent with that obtained by antibodies competing for binding to bins, and antibodies that cross-compete with each other for binding to HER-3 are also localized to the same domain on HER-3 (FIG. 3).

TABLE 4

Summary of monoclonal antibody binding domains based on ELISA assay results

XR ═ cross reaction-reaction

Example 9: determination of typical antibody classes

Chothia et al describe the structure of the hypervariable region of each immunoglobulin chain of an antibody according to the "canonical class" (J.mol.biol., 1987Aug 20, 196 (4): 901-17). A number of Fab and VL fragments of immunoglobulins were analyzed and the relationship of their amino acid sequences to the three-dimensional structure of their antigen binding sites determined. Chothia et al found that relatively few residues were primarily responsible for the backbone configuration of the hypervariable region by their stacking forces, hydrogen bonding or adopting the unusual phi, psi or omega configuration. These residues are often found at positions within the hypervariable regions, within the conserved β -sheet framework. By observing the sequences of immunoglobulins with unknown structures, Chothia et al indicate that many immunoglobulins have hypervariable regions of similar size to one of the known structures, and that they also contain identical residues at the sites responsible for the observed configuration.

Their findings indicate that these hypervariable regions have the same conformation as hypervariable regions of known structure. For the 5 hypervariable regions, the conformational library appeared to be limited to a relatively small number of discrete structural classes. These backbone conformations, which are frequently found in hypervariable regions, are referred to as "classical conformations". Chothia et al (Nature, 1989Dec 21-28, 342 (6252): 877-83) and others (Martin et al, J.mol.biol., 1996Nov 15, 263 (5): 800-15) demonstrated a small repertoire of at least five backbone conformations in six hypervariable regions of antibodies.

The CDRs of each of the antibodies described above were analyzed to determine their classical class. As is well known, the classical class has been assigned to the CDR1 and CDR2 of the antibody heavy chain along with the CDR1, CDR2 and CDR3 of the antibody light chain. The following table summarizes the results of the analysis. The classical class data is in the form of HCDR1-HCDR2-LCDR1-LCDR2-LCDR3, where "HCDR" refers to the heavy chain CDR and "LCDR" refers to the light chain CDR. Thus, for example, classical class 1-3-2-1-5 refers to an antibody having an HCDR1 belonging to classical class 1; one HCDR2, belonging to classical class 3; one LCDR1, belonging to classical category 2; one LCDR2, belonging to classical category 1; one LCDR3, belonging to classical category 5.

Antibodies with 70% or greater identity to the amino acids defining each classical class are assigned to a particular classical class. Amino acids defining each antibody can be found, for example, in the Chothia et al article referenced above. Classical class data for each HER-3 antibody are reported in tables 5and 6. When the identity is below 70%, the classical class classification is marked with an asterisk (") indicating that the best prediction of the appropriate classical class has been made based on the length of each CDR and the integrity of the data. When there are no classical classes of identical CDR length matches, the classical classes are labeled with the letter s and a number, such as "s 18," meaning that the size of the CDR is 18. When no sequence data is available for one heavy or light chain, the classical class is labeled with a "Z".

TABLE 5

Table 7 is an analysis of the amount of antibody per class. The number of antibodies with the particular canonical class indicated in the left column is shown in the right column. The four monoclonal antibodies lack one strand sequence data and therefore have a "Z" conformation in the typical classification, which is not included in the present calculation.

The most common structure is 3-1-2-1-1: of the 41 mabs containing the heavy and light chain sequences, 21 mabs had this combination.

TABLE 6

H1-H2-L1-L2-L3	Counting
		1-1-3-1-1	2
1-1-4-1^*-1	1
		1-2-2-1-1	4
1-2-8-1-1	1
		1-3-2-1-1	3
1-3-4-1-1	1
		3-1-2-1-1	21
3-1-4-1-1	5
		3-1-8-1-1	2
3-s 18-2-1-1	1

Example 10: determination of antibody affinity

The affinity of the anti-HER-3 antibodies of the invention was determined by indirect FACS Scatchard analysis. For this purpose, 10mM EDTA in PBS was used for collection of 10⁵Cells of interest or SK-Br 3 cells were washed with FAGS buffer (PBS, 3% FCS, 0.4% azide) and plated onto 96-well round-bottomed cell plates. The cells were centrifuged at 1000rpm for 3 minutes to remove the supernatant and then suspended either in the alpha-HER-3 antibody (3. mu.g/ml) or in any antibody dilution (100. mu.l/well) which started at 20. mu.g/ml and then diluted in a 1: 2 procedure. The cell suspension was incubated on ice for 1 hour, washed twice with FACS buffer, and then resuspended in donkey anti-human-PE (Jackson) secondary antibody (100. mu.l/well) diluted 1: 50 in FACS buffer. The cell suspension was incubated on ice and in the dark for 30 minutes, then washed twice with FAGS buffer and analyzed (FACS, BeckmanCoulter). According to FACS Scatchard scoreAnalysis, calculate the mean value of fluorescence for each measurement. Background staining (no primary antibody) was subtracted from each mean fluorescence. Scatchard plots were generated with x-value fluorescence mean and y-value fluorescence mean per mab (nM) concentration. KD is taken as the absolute value of 1/m of the linear equation. FIG. 4 shows kinetic analysis using the U1-59 antibody of the present invention. Table 8 below gives the affinity assays for certain antibodies of the invention selected using this method.

TABLE 7

Example 11: the anti-HER-3 antibody induces the endocytosis of HER-3 receptor

It has been determined that HER-3 can affect the development and progression of hyperproliferative diseases by acting as a "gatekeeper" of HER family-mediated cell signaling. Thus, if HER-3 is effectively cleared from the cell surface/membrane by receptor internalization, cell signaling and the resulting transition and/or maintenance of cells in the malignancy can eventually be abolished or prevented.

To investigate whether the anti-HER-3 antibodies of the invention could induce accelerated HER-3 endocytosis, the relative amounts of HER-3 molecules on the surface of cells treated with the anti-HER-3 antibodies of the invention for 0.5 and 4 hours were compared. 3X 10⁵Individual cells were seeded in normal medium in 24-well plates and allowed to grow overnight. Cells were preincubated with 10. mu.g/ml of anti-HER-3 monoclonal antibody in normal medium at 37 ℃ for the indicated time. The cells were detached with 10mM EDTA and incubated with 10. mu.g/ml of anti-HER-3 monoclonal antibody in washing buffer (PBS, 3% FCS, 0.04% azide) for 45 min at 4 ℃. Cells were washed twice with wash buffer, incubated with donkey anti-human-PE secondary antibody (Jackson) diluted 1: 100 for 45 min at 4 ℃ and analyzed by FACS (BeckmanCoulter, EXPO) after washing twice with wash buffer.

The data shown in FIG. 5 indicate that treatment of cells with HER-3 antibodies results in receptor internalization. Data are expressed as percent internalization and refer to the decrease in mean fluorescence intensity of anti-HER-3 treated samples relative to control treated samples.

Example 12: use of the human anti-HER-3 antibody of the invention for inhibiting ligands and human cancer cells Binding of SKBr3

Radioligand competition experiments were performed for quantifying the ability of an anti-HER-3 antibody of the invention to inhibit ligand binding to HER-3 in a cellular assay. For this purpose, the procedure for HER-3 receptor binding experiments used 4X 10⁵SK-BR-3 cells incubated with various concentrations of antibody on ice for 30 minutes. Then, 1.25nM [ alpha ] was added to each well¹²⁵I]-α-HRG/[¹²⁵I]β -HRG, incubated on ice for 2 hours. The microplate was washed 5 times, air dried and counted with a liquid scintillation counter. FIGS. 6a-e show the results of a manipulation experiment with a representative anti-HER-3 antibody of the invention, which indicates that the antibody of the invention is capable of specifically decreasing [ alpha ]¹²⁵I]-α-HRG/[¹²⁵I]Binding of β -HRG to cells expressing endogenous HER-3.

Example 13: inhibition of ligand-induced HER-3 phosphate by the anti-HER-3 antibodies of the invention Transforming

ELISA experiments were performed to investigate whether the antibodies of the invention are able to block ligand β -HRG mediated activation of HER-3. Ligand-mediated activation of HER-3 was detected by enhanced receptor tyrosine phosphorylation.

The first day: 1X 96 well plates were coated with 20. mu.g/mlI collagen in 0.1M acetic acid for 4 hours at 37 ℃. Inoculation 2.5X 10⁵The cells were cultured in normal medium.

The next day: cells were starved for 24 hours in 100 μ l serum-free medium.

And on the third day: cells were incubated with 10. mu.g/ml of anti-HER-3 monoclonal antibody for 1 hour at 37 ℃ in advance, and then with 30ng/ml of β -HRG-EGF domain (R)&D Systems) for 10 minutes. The medium was gently removed and the cells were fixed with PBS containing 4% formamide solution for 1 hour at room temperature. The formamide solution was removed and the cells were washed with washing buffer (PBS/0.1% Tween 20). With a content of 1% H₂O₂，0.1％NaN₃The cells were incubated at room temperature for 20 minutes and then blocked with NET-gelatine for 5 hours at 4 ℃. anti-phospho-HER-3 (Tyr1289) (polyclonal rabbit; Cell signaling # 4791; 1: 300) was added and allowed to act overnight at 4 ℃.

Day 4: the ELISA plate was washed 3 times with washing buffer, and then POD-labeled anti-rabbit antibody diluted 1: 3000 in PBS-0.5% BSA was added to each well for 1.5 hours at room temperature. The microplate was washed 3 times with washing buffer and once with PBS. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 650 nm. The reaction was stopped by adding 100. mu.l of 250nM HCl and reading the absorbance at 450nM using a Vmax microplate reader (Thermo Lab Systems) with 650nM as reference wavelength.

FIG. 7a shows representative results of this experiment, demonstrating that anti-HER-3 antibodies of the invention are capable of reducing ligand-mediated HER-3 activation, as evidenced by a reduction in receptor tyrosine phosphorylation. Data are expressed as percent reduction of therapeutic antibody relative to control antibody.

To determine the potential of mab U1-53 to inhibit ligand-induced HER-3 activation, MCF-7 cells were starved for 24 hours and incubated with mab U1-53 for 1 hour at 37 ℃ followed by 10 min stimulation with 10 nHRG-beta. Lysates were transferred to 1B4 (mouse anti-HER-3 monoclonal antibody) ELISA plates and assayed for HER phosphorylation using antibody 4G 10. As shown in FIG. 7b, phosphorylation of HER-3 was substantially completely inhibited and was dose dependent (IC50 at 0.14 nM).

Example 14: the human anti-HER-3 antibodies of the invention inhibit ligand induction p42/p44 MAP-kinaseEnzymatic phosphorylation

The ELISA experiments that were performed next were to investigate whether the antibodies of the invention could block ligand β -HRG mediated activation of p42/p44MAP kinase. Ligand-mediated activation of HER-3 was detected by enhanced protein phosphorylation (Thr202/Tyr 204).

The first day: 1X 96 well plates were coated with 0.1M acetic acid containing 20. mu.g/mlI type collagen at 37 ℃ for 4 hours. Inoculation 3X 10⁵The cells were cultured in normal medium.

The next day: cells were starved for 24 hours in 100 μ l serum-free medium.

And on the third day: cells were incubated with 5. mu.g/ml of anti-HER-3 monoclonal antibody for 1 hour at 37 ℃ in advance, and then with 20ng/ml of β -HRG-EGF domain (R)&D Systems) for 10 minutes. The medium was gently removed and the cells were fixed with PBS containing 4% formamide solution for 1 hour at room temperature. The formamide solution was removed and the cells were washed with wash buffer (PBS/0.1% Tween 20). With a content of 1% H₂O₂，0.1％NaN₃The cells were incubated at room temperature for 20 minutes and then blocked with PBS/0.5% BSA at 4 ℃ for 5 hours. A primary anti-phospho-p 44/p42MAP kinase (Thr202/Tyr204) (polyclonal rabbit; Cellsignaling # 9101; 1: 3000) was added and allowed to act overnight at 4 ℃.

Day 4: the ELISA plate was washed 3 times with washing buffer, and then HRP-labeled anti-rabbit antibody diluted 1: 5000 in PBS-0.5% BSA was added to each well for 1.5 hours at room temperature. The microplate was washed 3 times with washing buffer and once with PBS. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 650 nm. The reaction was stopped by adding 100. mu.l of 250nM HCl and reading the absorbance at 450nM using a Vmax microplate reader (Thermo Lab Systems) with 650nM as reference wavelength.

Fig. 8 shows representative results of this experiment. The anti-HER-3 antibodies of the invention are capable of reducing ligand-mediated activation of p42/p44MAP kinase, as evidenced by a reduction in phosphorylation. Data are expressed as percent reduction of therapeutic antibody relative to control antibody.

Example 15: inhibition of beta-HRG induction with the anti-HER-3 antibodies of the invention Phospho-AKT phosphorylation

In the following ELISA experiments, we investigated whether the anti-HER-3 antibodies of the invention block ligand β -HRG mediated activation of AKT kinase. Ligand-mediated AKT activation was detected with increased protein phosphorylation (Ser 473).

The first day: 1X 96 well plates were coated with 0.1M acetic acid containing 20. mu.g/ml type I collagen at 37 ℃ for 4 hours. Inoculation 3X 10⁵The cells were cultured in normal medium.

The next day: cells were starved for 24 hours in 100 μ l serum-free medium.

And on the third day: cells were incubated with 5. mu.g/ml of anti-HER-3 monoclonal antibody for 1 hour at 37 ℃ in advance, and then with 20ng/ml of β -HRG-EGF domain (R)&D Systems) for 10 minutes. The medium was gently removed and the cells were fixed with 4% formamide solution in PBS for 1 hour at room temperature. The formamide solution was removed and the cells were washed with washing buffer (PBS/0.1% Tween 20). With a content of 1% H₂O₂，0.1％NaN₃The cells were incubated at room temperature for 20 minutes and then blocked with PBS/0.5% BSA at 4 ℃ for 5 hours. Add primary anti-phospho-Akt (Ser473) (polyclonal rabbit; Cell signaling # 9217; 1: 1000) at 4 ℃ overnight.

The fourth day: the ELISA plate was washed 3 times with washing buffer, and then HRP-labeled anti-rabbit antibody diluted 1: 5000 in PBS-0.5% BSA was added to each well for 1.5 hours at room temperature. The microplate was washed 3 times with washing buffer and once with PBS. Tetramethylbenzidine (TMB, Calbiochem) was added and monitored at 650 nm. The reaction was stopped by adding 100. mu.l of 250nM HC1 and the absorbance at 450nM was read using a Vmax microplate reader (Thermo Lab Systems) with 650nM as the reference wavelength.

Fig. 9 shows representative results of this experiment. The anti-HER-3 antibodies of the invention are capable of reducing beta-HRG-mediated AKT, as evidenced by a reduction in phosphorylation. Data are expressed as percent reduction of therapeutic antibody relative to control antibody.

Example 16: inhibition of alpha-HRG/beta-HRG-mediated by anti-HER-3 antibodies of the invention MCF7 cell proliferation

In vitro experiments were performed to determine that the antibodies of the invention inhibit HRG-stimulated cell proliferation. 2000 MCF7 cells were seeded in 96-well plates containing FCS medium and cultured overnight. Cells were preincubated in quadruplicate with antibody diluted in medium containing 0.5% FCS at 37 ℃ for 1 hour. Direct addition of 30ng/ml α -or 20ng/ml β -HRG (R)&D Systems) ligand into antibody solution, and then allowing the cells to grow for 72 hours. Adding AlamarBlue^TM(BIOSOURCE) was allowed to act at 37 ℃ in the dark. Absorbance was measured at 590nm every 30 minutes. Data were recorded after adding AlamarBlue90 minutes. The results shown in fig. 10 indicate that representative antibodies of the invention inhibit HRG-induced growth of human cancer cells. Data are expressed as percent reduction of therapeutic antibody relative to control antibody.

Example 17: inhibition of beta-HRG-induced MCF7 cells with anti-HER-3 antibodies of the invention Cell migration

Migration experiments were performed to investigate whether the antibodies of the invention are able to block cell migration. The indicated amount of the antibody of the invention was added to the cell suspension of serum-starved MCF7 cells and pre-incubated at 37 ℃ for 45 minutes. Mu.l of cell suspension (50,000 cells) was placed in a collagen I coated transwells top chamber (BD Falcon, 8 μm pores). 750. mu.l of medium (MEM, amino acids, Na-pyruvate, Pen. -Strept, 0.1% BSA, no fetal bovine serum) without or with the ligand β -HRG-EGF domain (R & D Systems) was used in the bottom chamber. Cells were allowed to migrate at 37 ℃ for 8 hours and then stained with DAPI.

Stained nuclei were counted manually and percent inhibition was expressed relative to control antibody inhibition.

FIG. 11 shows the results of an experiment demonstrating that representative anti-HER-3 antibodies of the invention reduce HRG-induced cell migration.

Example 18: clone formation assay (Soft agar assay)

The soft agar assay was performed in order to investigate the ability of the anti-HER-3 antibodies of the invention to inhibit anchorage-independent cell growth. The soft agar colony formation assay is a standard in vitro assay for testing transformed cells, since only these transformed cells are able to grow on soft agar.

750 to 2000 wells (depending on the cell line) were preincubated for 30 min with 10. mu.g/ml of the indicator antibody in IMDM medium (Gibco) and then resuspended in 0.4% Difco purified agar (noble agar). The cell suspension was plated in quadruplicate on 0.75% agarose containing 20% FCS in the bottom layer of a 96-well plate. Colonies were allowed to form for 14 days and then stained overnight with 50. mu.l of 0.5mg/ml MTT in PBS. FIGS. 12a-i show the results of these experiments performed using representative antibodies in 3 of the present invention. The results indicate that the anti-HER-3 antibodies of the invention reduced growth of the following anchorage-independent cells: MDA-MB361 and NCI-ADR breast cancer cells (FIG. 12a, b), MKN-28 gastric cancer cells (FIG. 12c), HT144 melanoma cells (FIG. 12d), Skov3 ovarian cancer cells (FIG. 12e), PPC-1 prostate cancer cells (FIG. 12f), BX-PC3 pancreatic cancer cells (FIG. 12g), A431 epidermoid cancer cells (FIG. 12h), and lung cancer cells (FIG. 12 i). Clone counting was performed using a Scanalyzer HTS camera system (Lemnatec, Wuerselen).

Example 19: human anti-HER-3 antibodies inhibit the growth of human breast cancer in nude mice

Anti-tumor of therapeutic antibodiesTumor efficacy is often evaluated in studies of human transplanted tumors. In such studies, human tumors such as transplants were used to grow in immunocompromised mice and the efficacy of the antibody was measured by the degree to which tumor growth was inhibited. To determine whether the addition of anti-HER-3 antibody interfered with the growth of human breast cancer cells in nude mice, 5X 10 cells were used⁶One T47D cell was implanted into female NMRI nude/nude mice. Tumors were implanted subcutaneously and grown on the back of animals. After 8 days of tumor implantation, the mean volume reached 20mm³At this point, treatment is initiated. Before the first treatment, mice were randomly selected for statistical testing to ensure uniform tumor volume (mean, median and standard deviation) throughout the treatment group. Treatment was initiated with a first dose of 50mg/kg followed by intraperitoneal injections at a dose of 25mg/kg per week. One control forearm was injected with doxorubicin (pharmaceutical grade). All subjects were injected intraperitoneally with 0.5mg/kg of estrogen weekly.

Detailed information of the treatment groups is given below.

Doxorubicin therapy was described by Boven et al (Cancer Research, 1992).

The data for mean tumor volume (FIG. 13) show that the growth rate of the tumor is slowed down after administration of the anti-HER-3 antibody.

Example 20: human anti-HER-3 antibody inhibits human pancreatic tumors in SCID mice In (2) growth。

To test the potential therapeutic effect of anti-HER-3 antibodies on other types of solid tumor cells, tumors derived from the pancreatic tumor cell line BxPC3 were tested in mice with anti-HER-3 antibodies U1-53and U1-59. The control group of mice was treated with solvent control, PBS, or it was establishedThe therapeutic antibody Erbitux (Erbitux) of (1). 5X 10⁶BxPC3 cells (Matrigel-free) were subcutaneously transplanted into CB17 SCiD mice. Has a mean volume of 140mm²Tumor mice were injected intraperitoneally with 50mg/kgU1-53, U1-59, erbitux, or an equal volume of PBS. Thereafter, mice were injected with a weekly dose of 25mg/kg throughout the study.

The results of the experiment are shown in FIG. 14. U1-53and U1-59 reduced the growth rate of human pancreatic tumors by an inhibitory means. In this experiment, it is evident that U1-53and U1-59 are more effective than EGFR-targeting antibodies in delaying tumor growth. These data indicate that the therapeutic efficacy of the anti-HER-3 antibody is equivalent to a baseline therapeutic agent.

Example 21: human anti-HER-3 antibody binding to anti-EGFR antibody increases anti-tumor Activity of (2)

The use of targeted antibody monotherapy for hyperproliferative diseases is often hampered by problems such as the development of resistance on the one hand and antigenic changes on the other. For example, loss of antigenicity after prolonged treatment may render tumor cells insensitive to therapeutic antibodies, since those tumor cells that do not express or lose the targeted antigen have a selective growth advantage. These problems can be circumvented by using a therapeutic antibody that targets a different receptor of the tumor cell, or by using it with another anti-tumor drug. Intervention in multiple signaling pathways or even related pathways may provide therapeutic efficacy at multiple intervention levels. These combined treatments appear to be more effective because they combine two different anti-cancer drugs, each acting through a different mechanism of action.

To demonstrate the feasibility of the anti-HER-3 antibody, U1-53and U1-59 as suitable combination agents, we compared the effect of U1-53 or U1-59 monotherapy with U1-53 or U1-59 in combination with the anti-EGR specific antibody erbitux.5×10⁶A single BxPC3 cell (Matrigel-containing) was subcutaneously transplanted into CB17 SCID mice. When the tumor volume reaches 200mm³Thereafter, the mice were randomly divided into treatment groups. U1-53, U1-59and erbitux or anti-HER 3 antibodies were administered intraperitoneally alone or in combination with erbitux or as two anti-HER-3 antibodies weekly. The initial dose of all antibodies was 50mg/kg weekly, followed by 25mg/kg weekly for 6 weeks. The control group was dosed with gemcitabine (120mg/kg) bi-weekly, with weekly injections of mixed human IgG or solvent PBS. The protocol is as follows:

the results of the experiment are shown in FIG. 15. When antibodies U1-53 or U1-59 were administered alone, the growth of human pancreatic tumors was delayed to an equal extent as gemcitabine, which is commonly used as a standard drug for anti-pancreatic cancer chemotherapy. When U1-53 or U1-59 is administered with erbitux, tumor growth can be significantly reduced compared to U1-53, U1-59 or erbitux administered alone. Thus, by combining the anti-HER-3 antibody with a suitable targeting antibody unique to the tumor antigen, an effective therapeutic effect can be obtained.

In conclusion, anti-HER-3 antibodies have potent therapeutic effects on human tumors in vivo. Can be used together with other anti-tumor drugs to improve anti-tumor activity.

Example 22: nu/nu mice with human anti-HER-3 antibody inhibiting human melanoma In-growth

Members of the erbB family of receptors, including Her3, are not normally expressed in many epithelial tumors and play an important role in the growth and survival of many solid tumors. These solid tumors include melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and prostateCancer, glioma, gastric cancer, breast cancer, rectal cancer, pancreatic cancer, ovarian cancer. To determine that the anti-Her 3 antibodies of the invention are not limited to their anti-cancer activity against a single type of tumor (e.g. pancreatic cancer, example 21), but can also be used to treat a number of Her-3 dependent tumors, we additionally tested U1-53and U1-59 in a transplantation tumor experiment. An example is shown in figure 16. 5X 10⁵Each melanoma cell HT144 was injected subcutaneously into CB17 SCID mice, followed immediately by intraperitoneal injection of 50mg/kg of U1-53and U1-59, and an equal volume of PBS or 200mg/kg of Dacarbacin (DITC). Thereafter, mice received 25mg/kg of U1-53and U1-59 weekly, while DITC was administered at 200mg/kg bi-weekly.

Figure 16 shows the median tumor volumes from each panel. Administration of the antibodies of the invention resulted in reduced melanoma growth compared to tumors treated with control solutions. These results indicate that the antibodies of the invention are not limited to their therapeutic potential and can be targeted against a variety of cancers that express HER-3.

Example 23: human anti-HER-3 antibody inhibits the growth of colon cancer transplantable tumors in mice

HT-29 human colon cancer cells were suspended in 2: 1 with Matrigel in medium to a final concentration of 10X 10⁶One per ml. 0.2ml of cell suspension was injected subcutaneously into the right side of 4 to 5 week old CD1nu/nu mice. A total of 95 mice were used.

Mice were randomly selected and divided into control and treatment groups. Treatment was started on the same day. The treatment period lasted 29 days. After completion of the study, 3 tumors were collected 3 hours after each group was dosed. These tumors were snap frozen and placed at-80 ℃.

The operation is carried out according to the following processing method:

control group: nonspecific human IgG, 25mg/kg, twice weekly, was injected intraperitoneally

Treatment group: antibody U1-53, 25mg/kg twice weekly, intraperitoneal injection

Treatment group: antibody U1-7, 25mg/kg twice weekly, intraperitoneal injection

Treatment group: antibody U1-59, 25mg/kg twice weekly, intraperitoneal injection

5-FU treatment group: 5-fluorouracil, 50mg/kg, 9 dX 5, for intraperitoneal injection

Figure 17 shows the median tumor volumes from each panel. Administration of the antibody of the invention resulted in a reduction in the growth of colon cancer HT-29 compared to tumors treated with non-specific human IgG 1.

Example 24: human anti-HER-3 antibody inhibits lung cancer growth in mice

Calu-3 non-small cell lung cancer cells were mixed with Matrigel at a ratio of 1: 1, suspended in medium to a final concentration of 5X 10⁶Individual cells/ml. 0.05ml of cell suspension was injected subcutaneously into the right side of 9-week-old female CB17 scid mice. A total of 60 mice were used.

Mice were randomly selected and divided into control and treatment groups. Treatment was started on the same day. The treatment period lasted 32 days.

The operation is carried out according to the following processing method:

PBS group

hG control group: nonspecific human IgG: (25mg/kg), twice a week, intraperitoneal injection

Antibody U1-53 treatment group: 25mg/kg, twice a week, intraperitoneal injection

Antibody U1-7 treatment group: 25mg/kg, twice a week, intraperitoneal injection

Antibody U1-59 treatment group: 25mg/kg, twice a week, intraperitoneal injection

Figure 18 shows the median tumor volumes from each control and treatment group. Administration of the antibodies of the invention resulted in reduced growth of human non-small cell lung cancer transplantable tumors compared to tumors treated with PBS control or with non-specific human IgG.

Example 25: human anti-HER-3 inhibition of growth of human pancreatic cancer in Balb/C mice Long and long

Human pancreatic cancer BxPC3 tumor cells were suspended in the medium at 2: 1 with Matrigel to a final concentration of 5X 10 cells/cell⁶Individual cells/ml. 0.2ml of the cell suspension was injected subcutaneously into the right side of 5-7 week old female BalbC nu/nu mice. A total of 100 mice were used.

Mice were randomly divided into control and treatment groups. Treatment started on the same day for a total of 27 days.

The operation is carried out according to the following processing method:

control group of hIgG: nonspecific human IgG2, 25mg/kg, twice a week, i.p.

Treatment group: antibody U1-53, 25mg/kg, twice a week, intraperitoneal injection

Treatment group: antibody U1-7, 25mg/kg, twice a week, intraperitoneal injection

Treatment group: antibody U1-59, 25mg/kg, once a week, intraperitoneal injection

Gemzar treatment group: gemcitabine 80mg/kg, once a week, for intraperitoneal injection

Figure 19 shows the median tumor volumes from each control and treatment group. Administration of the antibodies of the invention resulted in reduced growth of human pancreatic cancer compared to tumors treated with non-specific human IgG or with Gemzar.

Inhibition of HER-3 in human pancreatic cancer is also shown in pharmacodynamic experiments. BxPC3 tumor grafts were grown as described above. 3 mice were treated with 500. mu.g of IgG1 control antibody and 3 mice were treated with anti-500. mu.g of HER-3 antibody U1-59. These mice were treated on the first and fourth days and then sacrificed on the fifth day to determine antibody-dependent HER-3 phosphorylation (pHER-3).

Tumors were homogenized in standard RIPA buffer plus protease inhibitors. 50 μ g of the clarified lysate was separated with 4-20% Tris-glycine gel, then transferred to nitrocellulose membrane and blocked with 3% Bovine Serum Albumin (BSA). Immunoblotting was performed using an anti-pHER-3 antibody (antibody 21D3, Cell Signaling technology). An anti-actin antibody (ABa-2066, Sigma) was used as a control.

Expression was detected using enhanced luminescence (Amersham Biosciences, Piscataway, N.J.). Images were captured with a Versadoc 5000 imaging system (BioRad, Hercules, CA).

Fig. 20 shows the result. After administration of the human anti-HER-3 antibody U1-59, phosphorylation of HER-3 could no longer be detected. Therefore, the antibody of the invention can obviously reduce the activation of HER-3 in human pancreatic cancer cells.

Example 26: use of the anti-HER-3 antibody of the invention as a diagnostic reagent

The anti-HER-3 monoclonal antibody can be used for diagnosing malignant tumor diseases. HER-3 is expressed in tumors in a very different manner compared to normal tissues, and therefore, analysis of HER-3 expression will help: preliminary diagnosis of solid tumors, pathological staging and histological grading of solid tumors, evaluation of prognostic criteria for hyperproliferative diseases and tumors, risk management of patients with HER-3 positive tumors.

A. Detection of HER-3 antigen in a sample

Enzyme-linked immunosorbent assay (ELISA) methods for the detection of HER-3 antigen in a sample have been developed. In this assay, wells of a microtiter plate (e.g., a 96-well microtiter plate or 384-well microtiter plate) are adsorbed for several hours by a fully human monoclonal antibody against the HER-3 antigen. This immobilized antibody is used to capture any HER-3 antigen that may be present in the test sample. All wells are washed and treated with blocking reagents such as milk protein or albumin to avoid non-specific adsorption in the analyte.

The wells of the microplate are then treated with a test sample suspected of containing the HER-3 antigen, or with a solution containing a standard amount of the HER-3 antigen. Such samples are, for example: a blood sample from which circulating HER-3 antigen is suspected to be contained in a subject is considered to be at a pathologically diagnostic level. After washing away the test sample or standard, the wells of the microplate are treated with a fully human anti-HER-3 secondary antibody of the invention conjugated with a biotin label. Labeled anti-HER-3 antibodies are used as detection antibodies. After washing away excess secondary antibody, the wells of the plate are treated with avidin conjugated to horseradish peroxidase (HRP) and a suitable chromogenic substrate. The concentration of the HER-3 antigen in the test sample is determined by comparison with a standard curve obtained from a standard sample.

B. Detection of HER-3 antigen in Immunohistochemistry (IHC)

To determine the HER3 antigen in tissue sections by immunohistochemistry, paraffin-embedded tissues were first deparaffinized twice with xylene for 5 minutes each, then dehydrated 2 times with 100% ethanol for 3 minutes each, dehydrated 1 minute with 95% ethanol, and rinsed with distilled water. Exposure to epitopes masked by formalin fixation and paraffin embedding can be resolved by epitope masking (epitopeumasking), enzymatic digestion or saponin. The epitope masking is removed by placing paraffin sections in an epitope recovering solution (e.g., 2N hydrochloric acid solution with pH 1.0), and heating in a steamer, water bath or microwave oven for 20-40 min. For enzymatic digestion, tissue sections are incubated in different enzyme solutions (e.g., proteinase K, trypsin, pronase, pepsin, etc.) for 10-30 minutes at 37 ℃.

After washing away the epitope retrieval solution or excess enzyme, the tissue sections were treated with blocking solution to prevent non-specific reactions. The primary antibody was diluted with the dilution buffer at the appropriate ratio and incubated at room temperature for 1 hour or overnight. Excess primary antibody was washed away and the sections were incubated in peroxidase blocking solution at room temperature for 10 minutes. After another washing step, the tissue sections are incubated with a secondary antibody, which is labeled with a group that can act as an enzyme anchor. An example is a biotin-labeled secondary antibody, which can be recognized by streptavidin conjugated with horseradish peroxidase. Detection of the antibody/enzyme complex may be achieved by incubation with a suitable luminescent substrate.

C. Determination of the concentration of HER-3 antigen in the serum of a patient

A sandwich ELISA method was established for quantifying HER-3 levels in human serum. Two fully human monoclonal anti-HER-3 antibodies are used in a sandwich ELISA method, which recognize different domains of the HER-3 molecule but do not compete for binding, e.g. see example 8. This ELISA was performed as follows: 50 μ l of anti-HER-3 coating antibody in coating solution (0.1M NaHCO)₃pH 9.6) was coated on a microplate (Fisher) at a concentration of 2. mu.g/ml. After overnight incubation at 4 ℃, the microplate was exposed to 200 μ l of blocking solution (0.5% BSA, 0.1% tween 20, 0.01% thimerosal, in PBS) for 1 hour at 25 ℃. The microplate was washed 3 times with PBS (Wash, WB) containing 0.05% Tween 20. Normal serum or patient serum (Clinomics, Biorecaimation) contained a 50% dilution of a blocking solution of human serum. The microplate was incubated with the serum sample overnight at 4 ℃, washed with a wash solution, and then incubated with a biotin-labeled anti-HER-3 detection antibody (100. mu.l/well) for 1 hour at 25 ℃. After washing, the microplate was incubated with horseradish peroxidase-labeled streptavidin for 15 minutes, washed as before, and then colored by reaction with o-phenylenediamine (Sigma developer) dissolved in hydrogen peroxide at 100. mu.l/well. The reaction was stopped with 2M sulfuric acid (50. mu.l/well) and analyzed at 492nm with a microplate reader. The concentration of HER-3 antigen in a serum sample was calculated by a 4-parameter curve fitting program compared to dilutions of purified HER-3 antigen.

Staging of cancer in a patient

Based on the results illustrated and discussed in items A, B and C, it was possible through the use of the present invention to stage cancer in a subject according to the expression level of the HER-3 antigen. In a given cancer type, blood samples are taken from subjects diagnosed at various stages in the development of the disease and/or at various time points of treatment. The concentration of the HER-3 antigen present in the blood sample is determined by a method that specifically measures the amount of antigen present. This method includes enzyme-linked immunosorbent assays (ELISA), such as the methods described in items A and B. A population of samples providing results statistically significant for each stage of development or treatment is used, specifying a range of concentrations of HER-3 antigen that can be considered characteristic for each stage.

To stage the progression of cancer in a subject under study, or to define the response of a subject to treatment, a blood sample from the subject is taken and the concentration of HER-3 antigen present in the sample is determined. The resulting concentration is used to determine in what concentration range the value decreases. The defined ranges correlate with the time period of development or treatment identified in the population of various diagnostic subjects, thus providing the time period of the subject under study.

Example 27: treatment or prophylaxis with the anti-HER-3 antibodies and antibody conjugates of the invention Prevention of hyperproliferative diseases

Many solid tumors are regulated by HER family-mediated signal transduction, and it has been demonstrated that HER-3 is a critical ligand that can form complexes with HER-1, HER-2 and HER-4. Thus, HER-3 mediated signal reduction or elimination will affect other HER family members and disrupt cellular signals, which brings about a broad therapeutic intervention and the potential to integrate therapy with other targeted drugs, organisms and cytotoxic drugs. Thus, the anti-HER-3 antibodies of the invention can be used to treat certain proliferative or HER-3 related disorders based on a number of factors, such as the expression of HER-3. Tumor types such as breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, rectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, other cancers in which HER-3 is expressed or overexpressed are currently preferred indications, but the indications are not limited to the preceding list. Furthermore, the following patient population would benefit from anti-HER-3 mab treatment.

Patients resistant to treatment with anti-HER-2 monoclonal antibodies

Patients not amenable to treatment with anti-HER-2 monoclonal antibodies

Patients resistant to anti-HER-1 monoclonal antibodies or small molecule anti-EGFR inhibitors

Small cell lung cancer patients with tolerance to erlotinib or gefitinib

The anti-HER-3 antibodies of the invention will be used in monotherapy or in combination with one or more pharmaceutical components in so-called "mixed therapy". The mixed therapy may include, but is not limited to, the agents defined by the signatures of the present invention. Combination therapy with anti-HER-3 antibodies and other agents can prolong patient survival and time to tumorigenesis or improve patient quality of life. The protocol and drug delivery design will describe the therapeutic effect and the ability to reduce the conventional dose of standard therapies (exemplified by chemotherapy and radiation therapy).

Treatment of humans with anti-HER-3 antibodies of the invention

To determine the in vivo efficacy of an anti-HER-3 antibody in treating a human tumor patient, the human patient is injected at a specific time with an effective amount of an anti-HER-3 antibody of the invention. During the treatment cycle, human patients are monitored to determine whether their tumors are progressing, in particular whether they are growing and spreading.

Tumor patients treated with the anti-HER-3 antibodies of the invention contain low levels of tumor growth and/or metastasis compared to the levels of tumor growth and spread in tumor patients treated with current standard of care.

Treatment with anti-HER-3 antibody conjugates of the invention

To determine the in vivo efficacy of the anti-HER-3 labeled antibody of the invention, a human patient or an animal exhibiting a tumor is injected at a specific time with an effective amount of the anti-HER-3 labeled antibody of the invention. For example, the administered anti-HER-3 labeled antibody is DM1 labeled anti-HER-3 labeled antibody, an auristatin labeled anti-HER-3 antibody, or an isotopically labeled anti-HER-3 antibody. During the treatment cycle, the human patient or animal is monitored to determine whether its tumor is developing, and in particular whether the tumor is growing and spreading.

Human patients or tumor-bearing animals treated with, for example, a DM 1-labeled anti-HER-3 antibody or an isotopically labeled anti-HER-3 antibody, contain lower levels of tumor growth and metastasis as compared to control patients or tumor-bearing animals treated by alternative methods. Control DM1 labeled antibodies that can be used in animals include a marker consisting of DM1 linked to an isotype antibody to an anti-HER-3 antibody of the invention, although in particular, the antibody does not have the ability to bind to a HER-3 tumor antigen. Control isotope-labeled antibodies that can be used in animal testing include markers consisting of isotopes that are linked to antibodies of the same type as the anti-HER-3 antibodies of the invention, although in particular the antibodies do not have the ability to bind to HER-3 tumor antigens. Note that: no control marker is administered to humans.

General description of the invention

The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the invention. The invention is not to be limited in scope by the recitation of the claims, as the recitation of an example is intended as a single recitation of specific objects of the invention, and any equivalent in function is within the scope of the invention. The material presented in the text does not constitute an admission that the written description contained in the text is insufficient to practice any of the objects of the invention, including the best mode thereof, nor does it limit the scope of the claims to their specific description.

The foregoing description and examples detail certain preferred embodiments of the invention, describing the best mode contemplated by the inventors. It should be understood, however, that no matter how detailed the foregoing appears in text, the invention may be practiced in many ways and the invention should be construed as being in accordance with the appended claims and their equivalents.

Furthermore, unless otherwise defined, all scientific and technical terms used in connection with the present invention shall have the meaning understood by those skilled in the art. Also, unless otherwise required by the context, singular terms shall include the plural and plural terms shall include the singular. In general, nomenclature and techniques used herein relating to cell and tissue culture, molecular biology, protein, oligonucleotide and polynucleotide chemistry, and hybridization are those well known and commonly employed in the art. Recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) are standard techniques used. The enzymatic reactions and purification techniques are performed according to manufacturer's instructions or routine procedures in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art or according to methods described in various conventional or special documents cited or discussed in this specification. See, for example, Sambrook et al, molecular cloning, A laboratory Manual (third edition, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by reference. The nomenclature used herein and the laboratory procedures and techniques related to analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

Merge of citations

All references cited herein, including patents, patent applications, articles, texts, etc., including references cited herein, are hereby incorporated by reference in their entirety.

Claims

1. An isolated antibody that binds to HER-3, characterized in that its heavy chain CDR1 consists of sequence GYTFTGYYMH, heavy chain CDR2 consists of sequence WINPNIGGTNCAQKFQG, heavy chain CDR3 consists of sequence GGRYSSSWSYYYYGMDV; and its light chain CDR1 is composed of the sequence KSSQSLLLSDGGTYLY, light chain CDR2 is composed of the sequence EVSNRFS, and light chain CDR3 is composed of the sequence MQSMQLPIT.

2. An isolated antibody that binds to HER-3, wherein: the heavy chain amino acid sequence is shown as SEQ ID NO: 42, and the light chain amino acid sequence thereof is set forth in SEQ ID NO: as shown at 44.

3. Use of an isolated antibody according to claim 1 or 2 for the preparation of a composition for binding the extracellular domain of HER-3.

4. Use of the isolated antibody of claim 1 or 2 in the manufacture of a composition for reducing HER-3 mediated signal transduction.

5. Use of the isolated antibody of claim 1 or 2 for the preparation of a composition for reducing phosphorylation of HER-3.

6. Use of an isolated antibody according to claim 1 or 2 in the preparation of a composition for reducing cell proliferation.

7. Use of the isolated antibody of claim 1 or 2 in the preparation of a composition for reducing cell migration.

8. Use of an isolated antibody according to claim 1 or 2 in the preparation of a composition for increasing the down-regulation of HER-3.

9. The isolated antibody of claim 1 or 2, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof, wherein the antibody fragment is a Fab fragment, a Fab 'fragment, F (ab')₂Fragments, Fv fragments, diabodies or single chain antibody molecules.

10. The isolated antibody of claim 9, wherein the isolated antibody is an IgG1-, IgG2-, IgG3-, or IgG 4-class.

11. The isolated antibody according to claim 1 or 2, wherein the isolated antibody is conjugated to a labeling group.

12. The isolated antibody of claim 11, wherein the labeling group is a radionuclide, a fluorescent group, an enzymatic group, a chemiluminescent group, or a biotin group.

13. The isolated antibody according to claim 1 or 2, wherein the antibody is conjugated to an effector group, wherein the effector group is a radionuclide, a toxin, or a therapeutic or chemotherapeutic group selected from the group consisting of calicheamicin, auristatin-PE, geldanamycin, maytansine and its derivatives DM 1.

14. An isolated nucleic acid molecule encoding the isolated antibody of claim 1 or 2.

15. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid molecule is operably linked to a control sequence.

16. A vector comprising the nucleic acid molecule of claim 14.

17. A vector comprising the nucleic acid molecule of claim 15.

18. A host cell transformed with the vector of claim 16.

19. A host cell transformed with the vector of claim 17.

20. A method of making the isolated antibody of claim 1 or 2, comprising the step of isolating the isolated antibody from a host cell.

21. The method of claim 20, wherein the host cell is a mammalian cell, a plant cell, a fungal cell, or a prokaryotic cell.

22. A pharmaceutical composition comprising the isolated antibody of claim 1 or 2, and a pharmaceutically acceptable carrier, diluent or adjuvant, wherein said antibody acts as an active agent.

23. Use of the pharmaceutical composition of claim 22 in the manufacture of a medicament for treating or preventing a hyperproliferative disease in a patient.

24. The use according to claim 23, wherein the hyperproliferative disease is selected from the group consisting of:

breast cancer, gastrointestinal cancer, pancreatic cancer, prostate cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, testicular cancer, soft tissue sarcoma, head and neck cancer, other cancers in which HER-3 is expressed or overexpressed, and the formation of tumor metastases.

25. The use according to claim 23 or 24, wherein the hyperproliferative disease is associated with an increased degree of HER-3 phosphorylation, an increased degree of HER-2/HER-3 heterodimerization or with PI₃Increased activity of-kinase, c-jun-terminal kinase, AKT, ERK2 and/or PYK 2.

26. A kit comprising the isolated antibody of claim 1 or 2.

27. The kit of claim 26, comprising a further therapeutic agent.

28. The kit of claim 27, wherein the further therapeutic agent is an antineoplastic agent.

29. The kit of claim 28 wherein the antineoplastic agent is an anti-tumor antibody or a chemotherapeutic agent.