KR101608301B1 - Antibodies Capable of Binding Specifically to HER2 - Google Patents

Abstract

The present invention relates to a novel HER2 antibody for use in combination therapy for cancer prevention or treatment. The antibody of the present invention is an antibody that specifically binds to HER2 that is overexpressed in cancer cells (particularly, breast cancer and gastric cancer cells), and is an antibody that binds to an epitope different from that of Trastuzumab. The antibodies of the present invention are very homologous compared to the CDR sequences of conventional HER2 target antibodies and have a unique sequence. The antibody of the present invention is highly effective in preventing or treating cancer (particularly, breast cancer and stomach cancer) by killing cancer cells by a greatly improved ability to kill cancer cells when administered in combination with trastuzumab. The superior efficacy of the conjugate administration by the antibody of the present invention can be achieved without wishing to be bound by theory that the antibody of the present invention binds to an epitope on HER2 different from that of trastuzumab, And cooperatively with HER2 inhibits HER2.

Description

Antibodies that specifically bind to HER2 {Antibodies Capable of Binding Specifically to HER2}

The present invention relates to a HER2 antibody used for the prevention or treatment of HER2 (Human Epidermal Growth Factor Receptor 2) related diseases, particularly cancer.

The HER2/neu (ErbB2) gene encodes a 185 kDa transmembrane glycoprotein, which is one of the family of epidermal growth factor receptors (EGFR). The HER2 protein is composed of an extracellular domain consisting of 620 amino acid residues, a transmembrane domain of 23 amino acid residues, and an intracellular domain of 490 amino acid residues having tyrosine kinase activity (Akiyama T, et al., Science , 232 ( 4758):1644-1646(1986)).

Meanwhile, HER2 antibodies having various properties have been disclosed in many literatures: Tagliabue et al., Int. J. Cancer 47:933-937 (1991); McKenzie et al., Oncogene 4:543-548 (1989); Maier et al., Cancer Res. 51:5361-5369 (1991); Bacus et al., Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al., PNAS (USA) 88:8691-8695 (1991); Bacus et al., Cancer Research 52:2580-2589 (1992); Xu et al., Int. J. Cancer 53:401-408 (1993); WO94/00136; Kasprzyk et al., Cancer Research 52:2771-2776 (1992); Hancock et al., Cancer Research. 51:4575-4580 (1991); Shawver et al., Cancer Res. 54:1367-1373 (1994); Arteaga et al., Cancer Res. 54:3758-3765 (1994); Harwerth et al., J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186; Kao et al., U.S. Publ. No. 2009/0285837 (2009); Ross et al., The Oncologist 8:307-325 (2003) and Klapper et al., Oncogene 14:2099-2109 (1997).

Among the HER2 antibodies, the most commercially successful antibody is trastuzumab antibody ( ^{commercialized as Herceptin TM} , US Pat. No. 5,821,337), which has been studied a lot: Sapino, A., et al., Annals of Oncology (2007) 18: 1963-1968; Bussolati, G, et al., British Journal of Cancer (2005) 92, 1261-1267; and Glazyrin A, et al., J Histology & Cytochemistry (2007) 55(1):25-33.

Although trastuzumab antibodies have been commercially successful, these antibodies tend to be effective only in about 15% of breast cancer patients with overexpression of HER2. Therefore, there are attempts to improve the prognosis of cancer patients who do not respond to trastuzumab or respond slightly to trastuzumab through concomitant administration in terms of the degree of efficacy or spectrum of efficacy (for drug-responsive cancer cell lines).

For example, US Patent Application Publication No. 2011-0086004 discloses an attempt to treat cancer by co-administration of a trastuzumab antibody and IL-21. US Patent Application Publication No. 2012-0107270 discloses a method of co-administering a substance obtained by conjugating IL-2 to a tenasine-C targeting antibody with a trastuzumab antibody.

US Patent Application Publication No. 2005-0101618 discloses a method of co-administration of a trastuzumab antibody and an erbB2 ligand. European Patent Application Publication No. 2134364 discloses a technique for inhibiting the proliferation of cancer cells by co-administration of a trastuzumab antibody and a telomerase inhibitor. WO 2008/031531 discloses a method of inhibiting cancer metastasis by co-administration of trastuzumab and pertuzumab.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have tried to develop antibodies capable of preventing or treating HER2-related diseases, particularly cancer (especially breast cancer and gastric cancer). In particular, the present inventors have tried to develop an antibody that can greatly overcome the limitations of anticancer activity that trastuzumab alone has when administered in combination with a trastuzumab antibody. As a result, the present inventors exhibited excellent binding ability to HER2 by themselves, and significantly improved efficacy in cancer (especially breast cancer and gastric cancer, more specifically HER2-expressing breast cancer and gastric cancer) by co-administration with trastuzumab. By developing a novel antibody that can be prevented or treated with, the present invention has been completed.

An object of the present invention is to provide an antibody or antigen-binding fragment thereof against HER2 (Human Epidermal Growth Factor Receptor 2).

Another object of the present invention is to provide a nucleic acid molecule encoding an antibody against HER2 or an antigen-binding fragment thereof.

Another object of the present invention is to provide a recombinant vector containing the nucleic acid molecule.

Another object of the present invention is to provide a host cell transformed with the recombinant vector.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.

Another object of the present invention is to provide a pharmaceutical composition for inducing apoptosis. Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to the first aspect of the present invention, the present invention provides a variable heavy chain comprising (a) CDR (complementarity determining region) H1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, and CDRH3 represented by the following general formula 1. It provides an antibody or antigen-binding fragment thereof against HER2 (Human Epidermal Growth Factor Receptor 2) comprising a region and (b) a light chain variable region:

General Formula 1

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ -Phe-Asp-Tyr

In the general formula, X ₁ is His, Asn, Ser or Ala, X ₂ is Leu, Phe, Tyr, His, Met, Trp, Asn, Ile or Ala, X ₃ is Gly or Cys, and X ₄ is Gly Or Ser, X ₅ is Thr, Met or Ala, X ₆ is Ala, Ser, Gly or Thr, and X ₇ is Ser, Ala, Cys or Thr.

The present inventors have tried to develop antibodies capable of preventing or treating HER2-related diseases, particularly cancer (especially breast cancer and gastric cancer). In particular, the present inventors have tried to develop an antibody that can greatly overcome the limitations of anticancer activity that trastuzumab alone has when administered in combination with a trastuzumab antibody. As a result, the present inventors exhibited excellent binding ability to HER2 by themselves, and significantly improved efficacy in cancer (especially breast cancer and gastric cancer, more specifically HER2-expressing breast cancer and gastric cancer) by co-administration with trastuzumab. A novel antibody that can be prevented or treated with has been developed.

The antibody of the present invention has a specific binding ability to HER2. In particular, the antibody of the present invention binds to an epitope different from the epitope to which trastuzumab binds, among the epitopes in HER2.

In the present specification, the term “trastuzumab” refers to an antibody disclosed in US Patent No. 5,821,337.

The antibody of the present invention has excellent killing ability or proliferation inhibitory ability against various HER2-expressing cancer cells. In the present specification, the terms "kill" or "proliferation inhibition" used while referring to cancer cells are used interchangeably with the same meaning.

In the present specification, the term “antibody” is a specific antibody against HER2, and includes not only the complete antibody form but also the antigen-binding fragment of the antibody molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma4(γ4), alpha1(α1) and alpha2(α2). The constant region of the light chain has kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)).

In the present specification, the term “antigen-binding fragment” refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab') ₂ and Fv. Among antibody fragments, Fab has a structure having a variable region of a light and heavy chain, a constant region of a light chain, and a first constant region of a heavy chain (C _H1 ), and has one antigen-binding site. F(ab') differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the _{heavy chain C H1 domain.} F(ab') ₂ antibodies are produced by disulfide bonds between cysteine residues in the hinge region of F(ab'). Fv is the smallest antibody fragment having only the variable region of the heavy chain and the variable region of the light chain, and the recombinant technology for generating the Fv fragment is PCT International Patent Application WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and It is disclosed in WO 88/09344. The double-chain Fv (two-chain Fv) is a non-covalent bond where the heavy chain variable region and the light chain variable region are connected, and the single-chain Fv (single-chain Fv) is generally shared by the variable region of the heavy chain and the variable region of the single chain through a peptide linker. It is connected by a bond or is directly connected at the C-terminus to form a dimer-like structure such as a double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the whole antibody with papain yields Fab, and digestion with pepsin yields F(ab') ₂ fragments), or It can be produced through gene recombination technology.

In the present invention, the antibody is preferably in the form of Fab or in the form of a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). Preferably, the constant region is gamma 1 (γ1), gamma 3 (γ3) and gamma 4 (γ4), most preferably gamma 1 (γ1) isotype. The light chain constant region may be of kappa or lambda type, preferably kappa type. Accordingly, a preferred antibody of the present invention is a Fab form or an IgG1 form having a kappa (κ) light chain and a gamma 1 (γ1) heavy chain.

In the present specification, the term “heavy chain” refers to a variable region domain V _H comprising an amino acid sequence having a sufficient variable region sequence for conferring specificity to an antigen and a whole including three constant region domains C _H1 , C _H2 and C _H3 It refers to both length heavy chains and fragments thereof. In addition, in the present specification, the term “light chain” refers to both a full-length light chain _{including a variable region domain V L} and a constant region domain C _L including an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen and fragments thereof. do.

As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the heavy and light immunoglobulin chains (Kabat et al., Sequences). of Proteins of Immunological Interest , 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy and light chains each contain three CDRs ((heavy chain (CDRH1, CDRH2 and CDRH3) and light chain (CDRL1, CDRL2 and CDRL3))), which provide the major contact residues for the antibody to bind to an antigen or epitope. do.

In the antibody of the present invention, CDRH3 is represented by general formula 1.

In General Formula 1, X ₁ is His, Asn, Ser or Ala, specifically His, Asn or Ser, more specifically His or Asn, and more specifically His.

In General Formula 1, X ₂ is Leu, Phe, Tyr, His, Met, Trp, Asn, Ile or Ala, specifically Leu, Phe, Tyr, His, Met, Trp, Asn or Ile, and more specifically Leu, Phe or Tyr, and more specifically Leu.

In General Formula 1, X ₃ is Gly or Cys, and specifically Gly.

In General Formula 1, X ₄ is Gly or Ser, specifically Gly.

In General Formula 1, X ₅ is Thr, Met or Ala, specifically Thr.

In General Formula 1, X ₆ is Ala, Ser, Gly, or Thr, and specifically Ala.

In General Formula 1, X ₇ is Ser, Ala, Cys or Thr, and specifically Ser.

According to an embodiment of the present invention, X ₁ is His, Asn or Ser, X ₂ is Leu, Phe or Tyr, X ₃ is Gly, X ₄ is Gly, and X ₅ is Thr, Met or Ala. , X ₆ is Ala, Ser, Gly or Thr, and X ₇ is Ser, Ala, Cys or Thr.

More specifically, X ₁ is His, Asn or Ser, X ₂ is Leu, Phe or Tyr, X ₃ is Gly, X ₄ is Gly, X ₅ is Thr, X ₆ is Ala, and X ₇ is Ser.

Even more specifically, CDRH3 includes any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27 to 28, SEQ ID NO: 32, and SEQ ID NO: 39 to 86, and even more Specifically, it includes the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 43, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 83, SEQ ID NO: 84 or 85, and most specifically It contains the amino acid sequence of the third sequence in the list.

According to an embodiment of the present invention, the light chain variable region includes CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 represented by the following general formula 2:

Formula 2

Y ₁ -Y ₂ -Y ₃ -Y ₄ -Y ₅ -Y ₆ -Pro-Trp-Thr

In the general formula, Y ₁ is Gln, Asp or Ala, Y ₂ is Gln, Asn, Glu or Ala, Y ₃ is Leu, Met, Asn, Ile, Ser, Thr, Ala or Lys, and Y ₄ is Tyr, Ala, Ser, Arg, Val, Gly, Met or Phe, Y ₅ is Ser, Phe, Tyr, Arg, Ile, Gly, Lys, Asn, Val or Ala, Y ₆ is Thr, Ser, Val, Ile, These are Ala, Gly, Asn, Glu, Phe and Leu.

In General Formula 2, Y ₁ is Gln, Asp or Ala, specifically Gln or Asp, and more specifically Gln.

In General Formula 2, Y ₂ is Gln, Asn, Glu or Ala, specifically Gln, Asn or Glu, and more specifically Gln.

In General Formula 2, Y ₃ is Leu, Met, Asn, Ile, Ser, Thr, Ala or Lys, specifically Leu, Met, Asn, Ile, Ser or Thr, and more specifically Leu, Met, Asn or Ile. , More specifically Leu or Met, and most specifically Leu.

In General Formula 2, Y ₄ is Tyr, Ala, Ser, Arg, Val, Gly, Met or Phe, and specifically Tyr or Ala.

In General Formula 2, Y ₅ is Ser, Phe, Tyr, Arg, Ile, Gly, Lys, Asn, Val or Ala, specifically Ser, Phe, Tyr, Arg or Ile, and more specifically Ser, Phe or Tyr. .

In General Formula 2, Y ₆ is Thr, Ser, Val, Ile, Ala, Gly, Asn, Glu, Phe and Leu, specifically Thr, Ser, Val, Ile, Ala, Gly or Asn, and more specifically Thr, Ser or Ala.

According to an embodiment of the present invention, Y ₁ is Gln or Asp, Y ₂ is Gln, Y ₃ is Leu, Met, Asn, Ile, Ser or Thr, and Y ₄ is Tyr, Ala, Ser, Arg, Val, Gly, Met or Phe, Y ₅ is Ser, Phe, Tyr, Arg or Ile, and Y ₆ is Thr, Ser, Val, Ile, Ala, Gly or Asn.

More specifically, the CDRL3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 33 to 38, and SEQ ID NO: 87 to SEQ ID NO: 245. SEQ ID NO: 6, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 178, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222 or SEQ ID NO: And, most specifically, the CDRL3 includes an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 88 (hz1E11-3), SEQ ID NO: 218 (hz1E11-133) or SEQ ID NO: 239 (hz1E11-154).

According to one embodiment of the present invention, the heavy chain variable region of the antibody of the present invention comprises an amino acid sequence of SEQ ID NO: 8 (1E11) or SEQ ID NO: 24 (hz1E11).

According to one embodiment of the present invention, the light chain variable region of the antibody of the present invention is SEQ ID NO: 10 (1E11), 26 (hz1E11), 247 (hz1E11-3), 249 (hz1E11-133) Or the amino acid sequence of SEQ ID NO: 251 (hz1E11-154).

As demonstrated in the examples below, the antibody of the present invention specifically binds to sub-domain 4 in the extracellular doamin (ECD) of the HER2 protein, but binds to an epitope different from trastuzumab.

The HER2 antibody or antigen-binding fragment thereof of the present invention may contain a variant of the amino acid sequence described in the attached sequence list within a range that can specifically recognize HER2. For example, changes can be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody.

These amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting an interactive biological function of a protein. It is a known fact that a similar biological activity can be retained only by substitution with an amino acid having a similar hydrophobicity index. When introducing a mutation with reference to the hydrophobicity index, substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equal biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0±1); Glutamate (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing a mutation with reference to a hydrophilicity value, substitution is made between amino acids showing a difference in hydrophilicity values of preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid exchanges in proteins that do not totally alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

Considering the above-described mutation having biologically equivalent activity, the antibody of the present invention or a nucleic acid molecule encoding the same is interpreted as including a sequence that exhibits substantial identity to the sequence listed in the sequence listing. The actual identity is at least 61% when the sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv . Appl . Math . 2:482 (1981); Needleman and Wunsch, J. Mol . Bio . 48:443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc . Acids Res . 16:10881-90 (1988); Huang et al., Comp . Appl . BioSci . 8:155-65 (1992) and Pearson et al., Meth . Mol . Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215:403-10(1990)) can be accessed from NBCI (National Center for Biological Information), etc. It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. The BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Antibodies of the present invention are monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-binding Fvs (sdFV) And anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies, but are not limited thereto.

On the other hand, the antibody CDR sequence of the present invention has very low similarity compared to the CDR sequences of conventional antibodies, and the sequence is unique. For example, the antibodies disclosed in U.S. Patent Nos.7329737 and 7993646, which were found to have the highest homology in the BLSAT search (http://www.ncbi.nlm.nih.gov/BLAST/), are the antibodies of the present invention. The 1E11 antibody, which is a parental antibody, has less than 50% homology in the total CDR sequence, and furthermore, the antibodies described in U.S. Patent Nos. 73273737 and 7993646 are antibodies that bind to hK-1, and the antibody of the present invention and its target are different. .

In conclusion, the amino acid sequence of the antibody of the present invention is recognized for its novelty and uniqueness.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention described above.

In the present specification, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugar or base moieties are modified. Also includes analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)). Nucleic acid molecule sequences encoding the heavy chain variable region and light chain variable region of the present invention may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.

According to one embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 7 or 23 in Sequence Listing.

According to one embodiment of the present invention, the nucleic acid molecule encoding the light chain variable region comprises a nucleotide sequence of SEQ ID NO: 9, 25, 246, 248, or 250 of Sequence Listing.

The nucleic acid molecule of the present invention encoding the HER2 antibody is interpreted as including a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. Homology of, more preferably at least 90% homology, and most preferably at least 95% of homology refers to a nucleotide sequence.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the above-described nucleic acid molecule.

As used herein, the term “vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cozmid vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors.

According to a preferred embodiment of the present invention, a nucleic acid molecule encoding a light chain variable region and a nucleic acid molecule encoding a heavy chain variable region in the vector of the present invention are operatively linked with a promoter.

In the present specification, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the The regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

For example, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., a metallotionine promoter, a β-actin promoter, a human heroglobin promoter, and Human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g. adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk Promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein Barr virus (EBV) promoter, and Loose sacoma virus (RSV) promoter) can be used, and transcription termination It generally has a polyadenylation sequence as a sequence.

The vector of the present invention may be fused with other sequences in order to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.

On the other hand, the expression vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are resistance genes for mycin and tetracycline.

According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector.

Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell can be used. For example, suitable eukaryotic host cells of the vector are monkey kidney cells 7 (COS7: monkey kidney cells), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells and HEK-293 cells Including, but not limited to.

According to another aspect of the present invention, the present invention provides the above-described (a) a pharmaceutically effective amount of the HER2 antibody or antigen-binding fragment thereof of the present invention; And (b) provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier.

Since the pharmaceutical composition of the present invention uses the above-described HER2 antibody or antigen-binding fragment thereof of the present invention as an active ingredient, the descriptions in common between the two are described in order to avoid excessive complexity of the present specification by repeated descriptions. Omit it.

As demonstrated in the following examples, when the HER2 antibody of the present invention is co-administered with trastuzumab, cancer cells (especially breast cancer cells, more specifically HER2-expressing breast cancer cells) are greatly improved in apoptotic activity. Since it can kill, it is very effective in the treatment of cancer (especially breast cancer and gastric cancer, more specifically HER2-expressing breast cancer and gastric cancer). According to one embodiment of the present invention, the pharmaceutical composition further comprises a trastuzumab antibody.

Cancers that can be prevented or treated by the composition of the present invention include various cancers known in the art, for example breast cancer, ovarian cancer, gastric cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer , Colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer or ureter cancer.

Specifically, cancers that can be prevented or treated by the composition of the present invention are HER2-expressing cancer, and more specifically, HER2-expressing breast cancer and gastric cancer.

According to another aspect of the present invention, the present invention provides (a) a pharmaceutically effective amount of the HER2 antibody or antigen-binding fragment thereof of the present invention; And (b) provides a pharmaceutical composition for inducing apoptosis comprising a pharmaceutically acceptable carrier.

According to one embodiment of the present invention, the pharmaceutical composition of the present invention is used for the prevention or treatment of hyperproliferative disease by inducing apoptosis, and the hyperproliferative disease is cancer, hyperplasia, keloid , Cushing syndrome, primary aldosteronism, erythroplakia, polycythemia vera, leukoplakia, hyperplastic scar, lichen planus, black. It is lentiginosis, arteriosclerosis, atherosclerosis, restenosis or stenosis.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered parenterally, such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The antibodies of the present invention can be used to diagnose, for example, diagnose HER2-expression related disorders, diseases or conditions.

According to another aspect of the present invention, the present invention provides a diagnostic kit for a disease, disease or condition related to HER2-expression comprising the antibody of the present invention described above.

The HER2-expression-related disease, disease or condition is specifically cancer, for example breast cancer, ovarian cancer, gastric cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer. , Prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer or ureter cancer. Specifically, the diagnostic kit of the present invention is used for diagnosis of HER2-expressing cancer, more specifically, HER2-expressing breast cancer and gastric cancer.

In addition, the antibody of the present invention can be used to analyze whether the antibody therapeutic agent of the present invention has drug responsiveness to a patient.

According to another aspect of the present invention, the present invention provides a drug responsiveness assay kit comprising the antibody of the present invention described above.

The assay kit of the present invention can be used to analyze whether a particular patient is medicinally responsive to the antibody of the present invention. For example, when a patient's cancer cells are treated with an antibody of the present invention, if a result is found that the antibody of the present invention binds, the patient can be determined to have drug reactivity with the antibody of the present invention.

Since the above-described kit contains an antibody, it can be basically made suitable for various immunoassays or immunostaining. The immunoassay or immunostaining includes radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence staining, and Including, but not limited to, immunoaffinity purification. The method of immunoassay or immunostaining is Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme- linked immunosorbent assay ( ELISA ), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out according to the radioimmunoassay method, ^{antibodies labeled with radioactive isotopes (eg, C 14} , I ¹²⁵ , P ³² and S ³⁵ ) detect HER2 receptors on the surface of cancer cells. Can be used to do. When the present invention is carried out by ELISA, a specific embodiment of the present invention comprises the steps of: (i) coating a sample to be analyzed on the surface of a solid substrate; (Ii) reacting the HER2 antibody as the primary antibody with the cell lysate; (Iii) reacting the result of step (ii) with a secondary antibody to which an enzyme is conjugated; And (iv) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes that catalyze a color development reaction, a fluorescence reaction, a luminescence reaction, or an infrared reaction, and examples include alkaline phosphatase, β-galactosidase, and hose. Radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) is used as a substrate. -B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when horse radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis -N-methylacridinium nitrate), resorupine benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS Substrates such as (phenzaine methosulfate) may be used.

When the present invention is carried out in a capture-ELISA method, a specific embodiment of the present invention comprises the steps of: (i) coating a HER2 antibody as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody and the sample; (Iii) reacting the result of step (ii) with a HER2 detecting antibody to which a label that generates a signal is bound; And (iv) measuring a signal generated from the label.

The detection antibody has a label that generates a detectable signal. The label includes chemicals (e.g., biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (e.g., C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), a fluorescent material (eg, fluorescein), a luminescent material, a chemiluminescent material, and a fluorescence resonance energy transfer (FRET), but are not limited thereto. For a variety of labels and labeling methods, see Ed Harlow and David Lane, Using Antibodies :A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

In the ELISA method and the capture-ELISA method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. If biotin is used as a label, streptavidin can be used, and when luciferase is used, a signal can be easily detected with luciferin.

Samples that can be applied to the kit of the present invention include, but are not limited to, cells, tissues or tissue-derived extracts, lysates or purified products, blood, plasma, serum, lymph or ascites.

The antibody of the present invention can be used for in vivo or in vitro imaging. According to another aspect of the present invention, the present invention provides a composition for an image comprising the antibody of the present invention described above and a label-coupled conjugate that generates a detectable signal bound to the antibody.

Labels that generate the detectable signal are T1 contrast material (eg, Gd chelate compound), T2 contrast material (eg, superparamagnetic material (eg, magnetite, Fe ₃ O ₄ ,γ-Fe ₂ O ₃ , manganese ferrite, cobalt). Ferrite and nickel ferrite)), radioactive isotopes (e.g. ¹¹ C, ¹⁵ O, ¹³ N, P ³² , S ³⁵ , ⁴⁴ Sc, ⁴⁵ Ti, ¹¹⁸ I, ¹³⁶ La, ¹⁹⁸ Tl, ²⁰⁰ Tl, ²⁰⁵ Bi and ²⁰⁶ Bi), fluorescent substances (fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5), chemiluminescent groups, magnetic particles, mass labels, or electron dense particles. It is not limited thereto.

The antibody of the present invention can be used alone to treat cancer, but since it can target HER2-expressing cells, it can be provided in the form of an ADC (antibody drug conjugate) by binding to other drugs.

Accordingly, according to another aspect of the present invention, the present invention provides an antibody pharmaceutical conjugate (ADC) comprising the antibody of the present invention and a drug bound to the antibody.

Drugs bound to the antibodies of the present invention are not particularly limited, and include chemicals, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents and enzyme inhibitors, and more specifically, anticancer agents such as: Acibicin, aclarubicin, acodazole, acronicin, adozelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutetimide, amonapide, ampligen, amsaclean , Androgens, Anguidin, Apidicholine Glycinate, Asarei, Asparaginase, 5-Azacytidine, Azathioprine, Bacillus Calmette-Guerine (BCG), Bakers Antipol, Beta-2 -Deoxythioguanosine, Bisanthrene HCl, Bleomycin Sulfate, Bulseophan, Butionine Sulfoximine, BWA773U82, BW 502U83/HCl, BW 7U85 Mesylate, Cerasemide, Carbetimer, Carboplatin, Carboplatin Mustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroid, corinerbacterium parvum, CPT-11, cristnatole, cyclocytidine , Cyclophosphamide, Cytarabine, Cytembena, Dabis Maleate, Decarbazine, Dactinomycin, Daunorubicin HCl, Diazauridine, Dexrazoic Acid, Deionhydrogalactitol, Diazicuone, Dibro Modulcitol, didemin B, diethyldithiocarbamate, diclicoaldehyde, dihydro-5-azacytine, doxorubicin, ethinomycin, dedatrexate, edelfosin, eflonitin, ellios solution , Elsamitrusin, Epirubicin, Esorubicin, Estramustine Phosphate, Estrogen, Ethanidazole, Ethiophos, Etoposide, Padrazole, Pazarabine, Penretinide, Pilgrastim, Finasteride, Flavone Acetic Acid , Phloxuridine, fludarabine phosphate, 5'-fluorouracil, Fluosol™, flutamide, gallium nitrate, gemcytabine, gosererin acetate, hefsulfam, hexamethylene bisacetamide, homoharingtonine, Hydrazine sulfate, 4-hydroxyandrostenedione, hydrodiurea, idaru Bicine HCl, ifosphamide, 4-ifomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisol, liposome daunorubicin, liposome capture doxorubicin, romastin, rhonidamine, mytansine, me Chloretamine hydrochloride, melphalan, menogaryl, merbaron, 6-mercaptopurine, mesna, methanol extract of Bacillus calete-guerine, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitoma Isine-C, mitotan, mitoxantrone hydrochloride, monosite/macrophage colony-stimulating factor, nabilon, napoxidine, neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, Pala, pentostatin, piperazindione, pipobroman, pyrarubicin, pyritrexim, pyroxantrone hydrochloride, PIXY-321, plicamycin, porpimer sodium, prednimustine, procarbazine, progestins, Pyrazofurine, Razox acid, Sargramostim, Semustine, Spirogermanium, Spiromustine, Streptonigreen, Streptozosine, Sulopener, Suramine Sodium, Tamoxifen, Taxorere, Tegapur, Teniposide, Terephthal Amidine, teroxilone, thioguanine, thiotepa, thymidine injection, thiazopurine, topotecan, toremifene, tretinoin, trifluperazine hydrochloride, trifluridine, trimetrexate, tumor necrosis factor (TNF) , Uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidin, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, melpharan, taxotel and taxol.

The features and advantages of the present invention are summarized as follows:

(a) The antibody of the present invention is an antibody that specifically binds to HER2 overexpressed in cancer cells (especially breast cancer and gastric cancer cells), and is an antibody that binds to an epitope different from trastuzumab.

(b) The antibody of the present invention has very low homology compared to the CDR sequences of conventional HER2 target antibodies, and the sequence is unique.

(c) When the antibody of the present invention is administered in combination with trastuzumab, it is very effective in preventing or treating cancer (especially breast cancer and gastric cancer) by killing cancer cells with greatly improved cancer cell killing ability.

(d) The excellence of the co-administration efficacy by the antibody of the present invention is bound to an epitope on HER2 different from that of trastuzumab, within a range without wishing to be bound by theory. By doing so, it is believed to inhibit HER2 cooperatively with trastuzumab.

(e) The antibody of the present invention can induce apoptosis, and thereby can be used for the prevention or treatment of hyperproliferative diseases.

(f) The antibody of the present invention can be used not only as a cancer therapeutic agent, but also as a diagnosis of cancer, analysis of drug reactivity, imaging, and ADC (antibody drug conjugate).

1 is a gene map of pcDNA3.3-IgG Heavy vector and pOptiVEC-IgG Kappa vector.
2A and 2B are graphs showing the efficacy of inhibiting proliferation by treatment with the 1E11 antibody alone and the combination treatment of the 1E11 antibody and trastuzumab against the NCI-N87 cancer cell line and the BT-474 cancer cell line, respectively. TRA, PER and hIgG represent trastuzumab, pertuzumab and human IgG (negative control), respectively.
3A-3B show that the 1E11 antibody binds to one site of HER2 sub-domain 4, which is different from the binding site of trastuzumab. 3A and 3B are SPR analysis results and ELISA analysis results, respectively.
4A is an ELISA result of analyzing whether the 1E11 antibody specifically binds to HER2 among ErbB family proteins to which HER2 belongs. CET (Cetuximab) was used as a control antibody against EGFR protein.
4B is an ELISA result of analyzing whether the 1E11 antibody binds to HER2 of a species other than human.
5A is a result of analyzing the ratio of cancer cells in which apoptosis occurred when 1E11 antibody and trastuzumab were treated alone or in combination with NCI-N87 cells.
5B is a result of analyzing the ratio of cancer cells in which apoptosis occurred when 1E11 antibody and trastuzumab were treated alone or in combination with BT-474 cells.
5C is a result of analyzing the cell viability after 24 hours and 48 hours of treatment with 1E11 antibody and trastuzumab alone or in combination with NCI-N87 cells. The control group represents the viability of cells treated with only PBS, which is an antibody solvent.
5D is a result of analyzing caspase-3/7 enzyme activity after 24 hours of treatment alone or in combination with 1E11 antibody and trastuzumab in NCI-N87 cells. The control group represents the viability of cells treated with only PBS, which is an antibody solvent.
6A is a Western blotting result showing the reduction of HER2 downstream signaling by treatment with 1E11 antibody and trastuzumab alone or in combination.
6B is a graph showing the inhibitory effect on heterodimerization-induced cell proliferation by treatment with 1E11 antibody and trastuzumab alone or in combination. Negative ctrl (negative control) is the survival rate of cells not treated with both ligand and antibody, and positive ctrl (positive control) is the survival rate of cells treated with only ligand and no antibody.
7A-7C show the effect of inhibiting tumor growth by treatment with 1E11 antibody and trastuzumab alone or in combination in an NCI-N87 xenograft tumor animal model. 7A is a graph showing a change in tumor volume, FIG. 7B is a graph showing a change in tumor weight, and FIG. 7C is an image showing a result of tumor tissue staining. Control Ab is parlivizumab.
Figures 8a and 8b are graphs showing the proliferation inhibitory efficacy by treatment of the humanized antibody hz1E11 antibody against the NCI-N87 cancer cell line and the OE-19 cancer cell line alone and the combination treatment of the hz1E11 antibody and trastuzumab, respectively.
9 shows the effect of inhibiting tumor growth by treatment with hz1E11 antibody and trastuzumab alone or in combination in an NCI-N87 xenograft tumor animal model. Control Ab is parlivizumab.
10A and 10B show alanine scanning results for CDRH3 and CDRL3 of the hz1E11 antibody, respectively.
Figures 11a-11f are affinity-enhanced humanized antibodies hz1E11-3, hz1E11-133 and hz1E11-154 for NCI-N87, OE-19 and BT-474 cancer cell lines alone and in combination with trastuzumab. It is a graph showing the effect of inhibiting proliferation.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Development of antibodies against HER2

For antibody development, an extracellular domain (ECD) site of HER2 protein was produced using animal cells, and then used as an antigen. The C-terminus of ECD was cloned into a pCEP4 vector (Invitrogen, Cat. No. V044-50) with a human IgG1 hinge and an Fc region (CH ₂ -CH _{3) bound using HindIII and BamHI restriction enzymes.} I did. Subsequently, ^{the cloned vector was transiently transformed into FreeStyle TM} 293F (Invitrogen, Cat. No. R790-07) cells using polyethyleneimine (Polyscience Inc., Cat. No. 23966), and a cell culture solution was performed. From, the HER2-ECD Fc fusion protein was purified using Protein-A Ceramic HyperD F resin (PALL, Cat No. 20078-028). The purified protein was quantified using Protein assay dye (Bio-Rad, Cat. No. 500-0006), and the concentration and purity were confirmed through Coomassie blue staining after SDS-PAGE. Purified 100 μg of protein antigen was mixed with Freund's adjuvant (Sigma, Cat. No. F5506), and injected into the abdominal cavity of BALB/c mice (Daehan Bio Co., Ltd.). After 2 weeks, 100 µg of the antigen was diluted in PBS for additional injection, and after 3 days, the spleen of the mouse was removed and lymphocytes were isolated. The isolated lymphocytes were mixed with Myeloma cell line SP2/0-Ag14 (ATCC, Cat. No. CRL-1581) at a ratio of 5:1, and fused using PEG-1500 (Roche, Cat. No. 783641). (fusion). The fused cells were cultured in a medium containing HAT supplement (Sigma, Cat. No. H0262), and the fused cells (hybridoma, hybridoma) were selectively selected and cultured.

The obtained hybridoma cells were confirmed to be cells producing antibodies that bind to antigens by ELISA method. HER2-ECD-Fc or ChromPure human IgG (hIgG, Jackson Immunoresearch Lab. Inc., Cat. No. 009-000-003) was added to a Costar 96-well plate (Corning, Cat. No. 3590) at a concentration of 1 μg/mL ) At room temperature for 1 hour. After washing three times with TBS-T (0.05% Triton X-100), it was blocked with 300 µl of TBS-T/SM (2% skim milk) for 30 minutes at room temperature. After washing the blocked plate 3 times, a hybridoma culture solution was added and the antibody was bound at 37° C. for 1 hour. After washing three times, anti-mouse IgG-HRP (Pierce, Cat. No. 31439) as a secondary antibody was diluted 1:5,000 in TBS-T/SM, and the antibody was bound at 37° C. for 1 hour. After washing three times, TMB (SurModics, Cat. No. TMBC-1000-01) was added and color was developed at room temperature for 5 minutes, and 1 N sulfuric acid (DukSan, Cat. No. 254) was added to stop the color development. Absorbance at 450 nm was measured using Victor X3 (PerkinElmer, Cat. No. 2030-0030), and antibodies that specifically bind to HER2-ECD-Fc were selected.

Since HER2 is a protein expressed on the cell surface, whether or not the developed antibodies bind to cells overexpressing HER2 was confirmed through cell-based ELISA. SKOV-3 (Korea Cell Line Bank, Cat. No. 30077), an ovarian cancer cell line that overexpresses HER2, was dispensed into a Costar 96-well cell culture plate (Corning, Cat. No. 3595) at 10,000 cells/well, and then 24 cells/well. Incubated for hours. The next day, the culture solution was removed, washed 3 times with PBS, and then the hybridoma cell culture solution was added, followed by further incubation at 37°C for 2 hours. After washing three times with TBS-T, goat anti-mouse IgG-HRP as a secondary antibody was diluted 1:5,000 in PBS/FBS (3% FBS), added, and treated at room temperature for 1 hour. After washing three times with TBS-T, color was developed using TMB. 61 clones showing absorbance higher than that using the negative control SP2/0 cell culture medium were selected.

Example 2: Comparison of the efficacy of the developed antibodies to inhibit breast cancer cell growth

Antibodies were purified from hybridoma cultures in order to perform a cell viability assay to confirm the breast cancer cell proliferation inhibitory effect. Hybridomas were cultured with a culture medium containing 3% FBS, and then an IgG-type antibody was purified using Protein-A resin. The purified antibody was quantified through BCA analysis (Pierce, Cat. No. 23227), and the concentration and purity were confirmed through Coomassie blue staining after SDS-PAGE.

Cell viability analysis was carried out by treatment alone or in combination with trastuzumab for BT-474, a typical breast cancer cell line overexpressing HER2, and NCI-N87, a gastric cancer cell line. In the case of combination treatment, the developed antibody and trastuzumab were mixed in a 1:1 ratio (weight ratio) and used. Dispense BT-474 (ATCC, Cat. No. HTB-20, 10,000 cells/well) and NCI-N87 (ATCC, Cat No. CRL-5822, 10,000 cells/well) cells in a 96-well plate for 24 hours. Cultured. The purified antibodies were treated to be 5 μg/mL, respectively, and BT-474 and NCI-N87 cell lines were further cultured for 4 days. In order to measure the viability of the cells, CCK-8 (Dojindo, Cat. No. CK-04-13) was added to a final 10%, and the absorbance was measured after treatment at 37° C. for 3 hours. The relative survival rate was calculated using the absorbance of the well not treated with the antibody as a survival rate of 100%. Through this, the 1E11 antibody was selected.

Example 3: Antibody sequence analysis

In order to analyze the antibody sequence, a phage Fab antibody library was prepared using RNA of each hybridoma, and from this, a screening process (panning) was performed three times to obtain phage binding to HER2-ECD-Fc (Phage display: a laboratory manual, Carlos Barbas III, et al., Cold Spring Harbor Laboratory Press). After hybridoma culture, RNA was isolated using the SV Total RNA Isolation System (Promega, Cat. No. Z3100), and cDNA was synthesized. Amplified the variable region of the antibody using a known primer set (see: Phage display: a laboratory manual, Carlos Barbas III, et al., Cold Spring Harbor Laboratory Press) and linked with human Ck and CH1 to pComb3X vector (Barbas laboratory, The Scripps Research Institute) using SfiI restriction enzyme, and transformed into ER2537 bacteria (New England Biolabs, Cat. No. 801-N). Transformed bacteria were infected with VCSM13 helper phage (Stratagene, Cat. No. 200251) to obtain phages and HER2-ECD-Fc-fixed immunotubes were used to obtain clones that bind to HER2-ECD-Fc.

Among the colonies of each antibody, an antibody binding to HER2-ECD-Fc was confirmed by ELISA. The transformed bacterial colonies were incubated at 37° C. so that the absorbance at 600 nm was 0.5, and then treated with IPTG to reach a final 1 mM, and cultured at 30° C. overnight to express the antibody in the form of Fab. After 5 mL culture, the cells were collected through centrifugation, and the cells were suspended in 0.4 mL of 1X TES (50 mM Tris, 1 mM EDTA, 20% (v/v) sucrose, pH 8.0), and then treated at 4° C. for 10 minutes. I did. 0.6 mL of 0.2X TES was added and further treated at 4° C. for 30 minutes, followed by centrifugation to take a supernatant. Costar 96-well half area plate (Corning Inc., Cat. No. 3690) coated with HER2-ECD-Fc at a concentration of 1 μg/mL was washed 3 times with TBS-T and then washed with TBS-T/SM (3% non -fat skim milk, 0.05% Triton X-100) was blocked at room temperature for 1 hour. The culture solution (Broth) or periplasmic extract (Periplasm) of each colony was diluted in a ratio of 1:3 using TBS-T/SM, treated, and then combined at room temperature for 1 hour. After washing three times, anti-HA-HRP (Roche, Cat. No. 120-138-190-01) was diluted 1:5000 as a secondary antibody and bound for 1 hour at room temperature. After washing three times, color was developed using TMB. .

Most colonies showed absorbance of 0.2 or higher in cell culture medium or periplasmic extract, and the nucleotide sequence of the antibody was analyzed for these clones. As a result of nucleotide sequence analysis, it was confirmed that colonies derived from a single hybridoma had the same sequence. The amino acid sequence of the CDR (complementarity determining region) of the 1E11 antibody is summarized in Table 1.

Amino acid sequence of CDR (complementarity determining region) of 1E11 antibody Light chain Heavy chain CDR1 LASQTIGTWLA SYTMS CDR2 ATSLAD YISNGGGSTYYPDTVKG CDR3 QQLYSTPWT HLGGTASFDY

Example 4: Preparation and production of chimeric antibodies

In order to make the antibody of the present invention in a more druggable form, a chimeric antibody was prepared.

Amplify the variable region of the mouse antibody that has been subjected to sequencing analysis _{, connect the human constant regions Cκ and C H} to the heavy chain portion, and clone the TA into pcDNA3.3-TOPO (Invitrogen, Cat. No., K8300-01) vector. The light chain portion was TA cloned into a pOptiVEC-TOPO (Invitrogen, Cat. No., 12744-017) vector. The primers used for amplification are shown in Tables 2 and 3, and a ClaI restriction site was added to the forward primer, and a NheI restriction site was added to the heavy chain and a BsiWI restriction enzyme site was added to the light chain, respectively. In addition, a signal sequence was added to the forward primer of the variable region so that the chimeric antibody could be secreted into the cell culture medium. _{The nucleotide sequence and amino acid sequence of Cκ and C H} used in the present invention are described in SEQ ID NOs: 11 to 14 of the Sequence Listing.

Using the above-described primers and GoTaq DNA polymerase (Promega, Cat. No. M3005), PCR reactions of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds were repeated 35 times. Each PCR product of the amplified variable region and constant region was subjected to electrophoresis on a 1% agarose gel, and then purified using a Qiaquick gel extraction kit (QIAGEN, Cat. No. 28706). To connect the variable region and the constant region, the PCR product of the variable region and the PCR product of the constant region are mixed in equal amounts, and then overlap extension PCR is performed using the forward primer of the variable region and the reverse primer of the constant region. Thus, a gene product was prepared and purified by the same method as above. The overlapping extension PCR was carried out by repeating the reaction 35 times at 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds using GoTaq DNA polymerase (Promega, Cat. No. M3005). The amplified gene product is in the pcDNA3.3-TOPO (Invitrogen, Cat. No., K8300-01) vector for the heavy chain portion and the pOptiVEC-TOPO (Invitrogen, Cat. No., 12744-017) vector for the light chain portion of the manufacturer. TA was cloned according to the manual.

Primers for variable region amplification Primer name order LF-1 CCG ATCGAT ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACGTGG GATATTCAGATG LR-1 CGG CGTACG TTTCAGCTCCAGCTTGG HF-1 CCG ATCGAT ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACGTGG GAGGTGAAGCT HR-1 CGG GCTAGC TGAGGAGACGGTGAC

Primer for constant region amplification Primer name order Ck-F GGAGCTGAAA CGTACG GTGGCTGCACC Ck-R CCG CTCGAG TTAACACTCTCCCCTGTTG CH-F CACCGTCTCCTCA GCTAGC ACCAAGGGCCCATCG CH-R CCG CTCGAG TCATTTACCCGGGGACAGGGAG

In the above table, the boldface indicates the restriction enzyme cleavage site, and the underline indicates the signal sequence.

A map of the finally prepared pcDNA3.3-IgG Heavy vector and pOptiVEC-IgG Kappa vector is shown in FIG. 1.

Then, ^{the cloned vector was transiently transformed into FreeStyle TM} 293F (Invitrogen, Cat. No. R790-07) animal cells using polyethyleneimine (Polyscience Inc., Cat. No. 23966), and Chimeric antibodies were purified from the cell culture medium using Protein-A Ceramic HyperD F resin (PALL, Cat No. 20078-028). The purified chimeric antibody was quantified using BCA analysis (Pierce, Cat. No. 23227), and the concentration and purity were confirmed through Coomassie blue staining after SDS-PAGE.

Example 5: Comparison of breast and gastric cancer growth inhibitory efficacy of the developed antibodies

In order to confirm the anticancer effect according to the concentration of the developed antibodies, cell viability analysis was performed on the breast cancer cell line BT-474 overexpressing HER2 and the gastric cancer cell line NCI-N87. BT-474 (10,000 cells/well) and NCI-N87 (10,000 cells/well) were dispensed into a 96-well plate in a volume of 70 μl, and cultured for 24 hours to fix. The next day, 30 μl of antibody was added to the cells in the culture. The final concentration of the treated antibody was at most 20 µg/mL for each antibody, and it was sequentially diluted 1:4 and proceeded at 5 concentrations. In the case of combined treatment with trastuzumab, the developed antibody and trastuzumab were in a 1:1 ratio (for example, in FIGS. 2A and 2B, when the dose is 1 μg/mL, TRA 1 μg/mL and 1E11 1 Μg/mL was administered). After antibody treatment, BT-474 and NCI-N87 cells were further cultured for 4 days, and then CCK-8 was added to a final 10%, followed by treatment at 37° C. for 3 hours. Then, the absorbance was measured at 450 nm using Victor X-3. The absorbance of the cells not treated with the antibody was set to 100%, and the relative viability was calculated (FIGS. 2A and 2B ).

The developed 1E11 antibody showed proliferation inhibitory efficacy against NCI-N87 (FIG. 2A) and BT-474 (FIG. 2B) cell lines that respond to trastuzumab. Moreover, the 1E11 antibody exhibited superior cancer cell proliferation inhibitory efficacy against NCI-N87 and BT-474 cell lines compared to trastuzumab alone when treated in combination with trastuzumab. Interestingly, the combination treatment of 1E11 antibody and trastuzumab showed superior proliferation inhibitory efficacy for the NCI-N87 cell line than the combination treatment of trastuzumab and pertuzumab (FIG. 2A).

Example 6: of the developed antibody Trastzumab Synergy effect verification in parallel processing

In order to analyze whether the anticancer efficacy seen when administered concurrently with trastzumab in gastric cancer of the developed 1E11 antibody is a synergistic effect, the anticancer efficacy shown by treatment with 1E11 antibody and trastuzumab alone or in combination on NCI-N87 cells was analyzed ( Fig. 2a). The anticancer efficacy according to the concentration was determined by the CalcuSyn program (Biosoft) using the Chou & Talalay method (Chou et al., Adv. Enzyme. Regul. 22:27-55 (1984)) to analyze the co-administration efficacy of two or more drugs. Was analyzed using (Table 4). When the two drugs are co-treated, the two drugs are agonistic, additive, or synergistic. Drug interactions using the Chou & Talalay method can be analyzed by CI (combination index). If the CI values of both drugs are 1 or more, it means agonistic, if it is 1, it shows additive, and if it is less than 1, it shows synergistic effect.

1E11 + Trastzumab C.I. r ED ₅₀ ED ₇₅ ED ₉₀ 0.0315 0.0459 0.0751 0.95921

In the above table, ED ₅₀ , ED ₇₅ and ED ₉₀ denote effective doses showing an effect in 50%, 75% and 90% populations, respectively. r represents the linear correlation coefficient of the median-effect plot.

As can be seen in Figure 2a and Table 4, when the combination treatment of trastuzumab and 1E11 clone, the CI value of the efficacy of the two drugs is 0.1 or less, so it can be seen that these two antibodies show a strong synergistic effect when treated in combination.

Example 7: Comparison of the developed antibody with trastuzumab Epitope compare

The antibody trastuzumab against HER2 is known to bind to domain-4 of the four domains of the HER2 ECD. In order to check whether the developed antibodies overlap with the epitope for HER2 of trastuzumab, epitope binning was performed using a surface plasmon resonance (SPR) method using a Biacore 3000 (GE Healthcare) instrument. Trastuzumab was fixed to a CM5 sensor chip (GE Healthcare, Cat. No. BR-1000-12) by an amine coupling method using ECD/NHS to about 1,000 RU (response unit). HER2-ECD-His protein at a concentration of 320 nM 4 using HBS-P buffer (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.005% Tween-20, pH 7.4) on the sensor chip fixed with trastuzumab. The binding between trastuzumab and HER2-ECD was stabilized by binding for a minute and then flowing only a buffer for 5 minutes. Thereafter, the secondary antibody was bound at a concentration of 1 μg/mL for 4 minutes, and then a buffer was flowed. In all experimental procedures, the flow rate was used at 50 μl/min. If the secondary bound antibody further binds to the HER2-ECD protein bound to trastuzumab, it is an antibody that does not share an epitope with trastuzumab.

As can be seen in Fig. 3a, since hIgG used as a secondary antibody does not bind to HER2, there is no additional binding, and trastuzumab has the same epitope, so it does not bind further. On the other hand, 1E11 antibody was confirmed to have a different epitope than trastuzumab because it further bound to HER2-ECD bound to trastuzumab.

In order to identify the domain site to which the 1E11 clone binds, the four sub-domains (Domain1-4) constituting the extracellular domain of the HER2 protein were individually combined with the hinge and the Fc site of human IgG1 using animal cells. Produced and purified using Protein-A. The binding of the 1E11 clone, trastuzumab, and pertuzumab to the produced recombinant protein was confirmed by ELISA method. As can be seen in Figure 3b, it can be seen that the 1E11 clone is defective in sub-domain 4 in the same way as trastuzumab.

The above results indicate that the 1E11 clone binds to sub-domain 4 of the ECD of the HER2 protein, but binds to an epitope different from trastuzumab (FIG. 3).

Example 8: of the developed antibody HER2 Specificity for

It was confirmed by ELISA whether the developed 1E11 antibody specifically binds to HER2 among ErbB family proteins to which HER2 belongs, or to HER2 of a species other than human. To confirm whether the developed antibody specifically binds to HER2 among ErbB family proteins, the extracellular domains of EGFR, HER2, HER3 and HER4 belonging to the ErbB family were identified by ELISA. The extracellular domain of EGFR (EGFR-ECD-Fc) was produced in the same way as HER2-ECD-Fc, and HER3 (R&D Systems, #348-RB-050) and HER4 (R&D Systems, #1131-ER-050) Protein was purchased and used. In order to confirm whether the developed antibody exhibits cross-species cross-reaction with the HER2 protein of other species, human, rhesus monkey, cynomolgus monkey, mouse and rat HER2 cell external domains were identified. It was confirmed by the ELISA method used. The HER2 extracellular domain of cynomolgus monkey was produced in the same way as human HER2-ECD-Fc, HER2 of rhesus monkey (Sino Biological Inc., #90020-K08H), HER2 of mouse (Sino Biological Inc., #50714) -M08H), rat HER2 (Sino Biological Inc., #80079-R08H) extracellular domain was purchased and used.

As can be seen in Figures 4a and 4b, the developed 1E11 antibody specifically binds to HER2 among human ErbB family proteins, and it can be confirmed that there is a cross-species reaction with HER2 of rhesus monkeys and crabs.

Example 9: Efficacy in inducing apoptosis of the developed antibody

In order to analyze the mechanism of action of the anticancer efficacy seen when the treatment with trastuzumab and trastuzumab for gastric cancer and breast cancer overexpressing HER2 of 1E11 antibody, the apoptosis induction efficacy was analyzed. In order to analyze the apoptosis induction efficacy, 1E11 antibody and trastuzumab were treated in NCI-N87 and BT-474 cells at a concentration of 10 μg/mL alone or in combination for 48 hours (when combined treatment, trastuzumab 10 μg/ mL and 10 μg/mL of 1E11 were administered). After dropping the antibody-treated cells using trypsin enzyme, 500,000 cells were analyzed by flow cytometry (Cytomics FC500, Beckman Coulter Inc.) using the ApoScreen Annexin V Apoptosis kit (SouthernBiotech, #10010-02) (Fig. 5a and 5b).

In order to measure the enzymatic activities of caspase-3 and caspase-7, which are important enzymes involved in apoptosis, first, the anticancer efficacy over time was analyzed when 1E11 and trastuzumab were treated alone or in combination. NCI-N87 cells were treated with an antibody at a concentration of 10 μg/mL, and cell viability after 24 hours and 48 hours was analyzed using a CellTiter-Glo (Promega, #G7571) reagent (FIG. 5C). As a result, it was confirmed that the cell viability rapidly decreased after 24 hours. Based on these experimental results, the enzyme activity of caspase-3/7 was confirmed 24 hours after antibody treatment. In order to measure caspase-3/7 enzyme activity, NCI-N87 cells were treated with an antibody at a concentration of 10 μg/mL for 24 hours and analyzed using a Caspase-Glo 3/7 Assay (Promega, #G8091) kit. (Fig. 5d).

As can be seen in Figures 5a and 5b, it can be seen that the 1E11 antibody alone has an apoptosis induction effect different from trastuzumab alone against HER2 overexpressing gastric cancer (NCI-N87 cells) and breast cancer (BT-474 cells). It can be seen that such apoptosis efficacy is further increased when combined with trastuzumab. The efficacy of inducing apoptosis according to the combination treatment of 1E11 and trastuzumab can be reconfirmed by increasing the activity of caspase-3/7, an enzyme important for apoptosis (FIG. 5D).

Example 10: of the developed antibody HER2 Cell signaling inhibitory effect

In order to analyze the mechanism of action of the anticancer efficacy seen when the 1E11 clone was combined with trastuzumab for gastric cancer and breast cancer overexpressing HER2, the inhibitory effect of HER2 on the intracellular signaling system was analyzed. Antibodies were treated with NCI-N87 cells at a concentration of 10 μg/mL for 24 hours. Cell extract solution from antibody-treated cells (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM NaF, 1 mM Na ₃ VO _{4 ,} 1 mM PMSF and protease Inhibitor cocktail (Sigma)) was used to obtain a cell extract. The cell extract was analyzed using a special protein detection test (Western blot). For analysis, Cell Signaling Technology's HER2 (#4290), pHER2 (#2243), pHER3 (#4791), EGFR (#4267), pEGFR (#3777), AKT (#4691), pAKT (#4060), ERK (#4695) and pERK (#4370) antibodies, and HER3 (sc-285) antibodies manufactured by Santa Cruz Biotechnology were used. GAPDH (AbClon, #Abc-1001) was used as a loading control. Each protein was visualized in line form using HRP-conjugated anti-mouse antibody (AbClon, AbC-5001), anti-rat antibody (AbClon, AbC-5003) and AbSignal (AbClon, #AbC-3001) reagents.

It was confirmed whether HER2 inhibits heterodimerization with EGFR or HER3, which are proteins in the other ErbB family, by combined treatment with the developed 1E11 antibody and trastuzumab. EGF was treated to induce heterodimerization of HER2 and EGFR, and HRG was treated to induce heterodimerization of HER2 and HER3. NCI-N87 cells were aliquoted using a cell culture solution containing 0.1% FBS and cultured for 24 hours. First, the antibody was treated at a concentration of 10 μg/mL for 1 hour, and then EGF (R&D Systems, #236-EG-200) or HRG (R&D Systems, #377-HB/CF) was treated at a concentration of 200 ng/mL. Cell viability was analyzed after 3 days.

As can be seen in Figure 6a, it was confirmed that the HER2 protein was reduced when 1E11 and trastuzumab were co-treated. As the HER2 protein decreased, the phosphorylated HER2 protein also decreased. In the case of EGFR and HER3, which are known to be heterodimerized with HER2, there is no change in the amount of protein, but it can be confirmed that the amount of phosphorylated protein decreases. This means that when 1E11 and trastuzumab are co-treated, the activities of HER2 and EGFR and HER3 can be regulated. Through this regulation, it was confirmed that AKT and ERK, which are known as sub-cell signaling systems of HER2, are not quantitatively regulated but their activity is regulated.

6B, it can be seen that cell proliferation through heterodimerization of HER2 with EGFR or HER3 decreases when 1E11 and trastuzumab are co-treated. Cell proliferation of NCI-N87 by EGF, which induces heterodimerization of HER2 and EGFR, and HRG, which induces heterodimerization of HER2 and HER3, is combined with 1E11 and trastuzumab, where HER2 binds to other receptors. It can be seen that it suppresses the similar degree to that of Pertuzumab, which is known to inhibit it.

The above results indicate that when 1E11 and trastuzumab are co-treated, HER2 inhibits cell signaling transmitted through heterodimerization with EGFR and HER3.

Example 11: Analysis of anti-cancer efficacy using an animal model of the developed antibody

The anticancer efficacy of 1E11 was verified using an animal model. A xenograft model was used to generate cancer by subcutaneous injection of 5,000,000 NCI-N87 cells into a thymic nude female mouse (Daehan Biolink). The animals were randomly divided into 4 groups when the cancer grew to a size of about 200 mm ^3. The antibody developed in each group of mice and the isotype control of trastuzumab were parlivizumab (MedImmune LLC.), 1E11, trastuzumab, and 1E11 and trastuzumab at 10 mg/kg, respectively. It was injected intraperitoneally twice a week (intraperitoneal administration). In the case of co-administration, each antibody was administered at a dose of 10 mg/kg. During the experiment, the volume of the cancer tissue was measured, and on the 22nd day of antibody administration, the animal was sacrificed to extract the cancer tissue. The volume of the cancer tissue was calculated by the equation (L*W*W)/2, and the longest diameter of the measured diameters is L and the shortest diameter is W. The extracted cancer tissue was weighed and used for tissue staining. Tissue staining was fixed with formalin and paratin-embedded section followed by H&E (hematoxylin (DAKO, #CS700); eosin (DAKO, #CS701)) and an antibody against HER2 (Cell Signaling Technology, #4290).

It can be seen that when 1E11 is administered alone, cancer growth is reduced to a level similar to that of trastuzumab (FIG. 7A). When 1E11 was co-administered with trastuzumab, it could be confirmed that cancer growth was significantly suppressed compared to when each antibody was administered alone. The cancer growth inhibitory effect was also confirmed by the difference in weight of the cancer tissue extracted after performing the experiment (FIG. 7B). Through tissue staining, when 1E11 and trastuzumab were co-administered, cells with reduced HER2 expression levels were confirmed as a result of confirming in a special protein detection test (Western blot) method (FIG. 6A) (FIG. 7C). The above results indicate that the combination of 1E11 and trastuzumab inhibits cancer growth more than that of single administration, and one of the mechanisms of inhibition is through inhibition of the expression of HER2 protein.

Example 12: Humanized antibody development and efficacy verification

A humanized antibody of the chimeric 1E11 antibody developed in Example 4 was developed using a CDR grafting method. As for the human antibody to be transplanted with the CDR of the developed antibody, the V and J genes of the human germline antibody gene with high nucleotide sequence similarity were selected using IMGT/V-QUEST (Brochet, X. et al., Nucl. Acids Res. 36:503-508 (2008)). IGHV3-48*03 and IGHJ4*01 genes were selected as the V and J genes of the heavy chain, and their similarities were 85.07% and 87.23%, respectively. In addition, IGKV1-39*01 and IGKJ1*01 genes were selected as the V and J genes of the light chain, and their similarities were 81.36% and 81.08%, respectively. Alanine instead of serine in human germline genes according to Kabat numbering criterion H49 of the heavy chain corresponding to the Vennier zone, which can affect the overall structure of the antibody, because it has been reported that affinity is reduced when only CDR is transplanted. Replaced with. The developed humanized antibody hz1E11 was produced in the form of IgG ^{using the FreeStyle TM} 293F cell line. The amino acid sequences of the developed heavy chain variable region and light chain variable region of hz1E11 are described in SEQ ID NOs: 24 and 26, respectively.

The affinity of the developed humanized antibody hz1E11 of 1E11 to HER2 was measured using a surface plasmon resonance (SPR) assay. All experiments were conducted using a Biacore 3000 instrument. First, an antibody against human IgG of goat was fixed to a CM5 sensor chip at 1000 RU by an amine coupling method using ECD/NHS. Trastuzumab was diluted to 2.84, pertuzumab was 5.68, and hz1E11 was 7.1 using HBS-P buffer. Before binding of the HER2-ECD-His protein, each antibody was bound at a rate of 50 μl/min for 180 seconds, and a buffer was flowed for 5 minutes to stabilize. Thereafter, the HER2-ECD-His protein was bound at a concentration of 640 nM, 320 nM, 160 nM, 80 nM, 40 nM, 20 nM, and 0 nM for 4 minutes, and a buffer was flowed for 15 minutes. The sensor chip was regenerated by flowing 10 mM Glycine-HCl (pH 1.5) buffer for 15 seconds. All sensorgram data were analyzed by 1:1 interaction model using BIAevaluation software. The affinity of the antibody is summarized in Table 5. The affinity of trastuzumab and pertuzumab was 1.94 nM and 1.89 nM, respectively, while the affinity of the developed antibody 1E11 was 16.0 nM. It was confirmed that the affinity of the humanized antibody of 1E11 is 10.4 nM, which is almost no difference from the existing 1E11 antibody.

Antibody K _a (M ^-1 s ^-1 ) K _d (s-1) K _D (nM) Trastuzumab 3.9E+04 7.6E-05 1.94 Pertuzumab 3.6E+04 6.8E-05 1.89 1E11 3.0E+04 4.7E-04 16.0 hz1E11 4.9E+04 5.1E-04 10.4

The anti-cancer efficacy of the developed 1E11 humanized antibody hz1E11 was verified in the NCI-N87 and OE-19 cell lines, which are human gastric cancer cells overexpressing HER2 (FIG. 8). In NCI-N87 cells, when hz1E11 was treated alone, the cancer survival rate decreased to a level similar to that of trastuzumab, and when hz1E11 was treated in combination with trastuzumab, it was confirmed that the survival rate of cancer significantly decreased compared to the single treatment of each antibody. (Fig. 8a). In OE-19, which is another HER2 overexpressing human gastric cancer cell line, it was confirmed that the cancer survival rate was decreased when the hz1E11 and trastuzumab were also treated in combination compared to the single treatment of each antibody (FIG. 8B). In addition, the combination treatment with hz1E11 and trastuzumab for NCI-N87 showed a slightly better inhibitory effect on cancer cell proliferation than the combination treatment with 1E11 and trastuzumab (FIG. 2A) (FIG. 8A). The combination treatment of hz1E11 antibody and trastuzumab showed superior cancer cell proliferation inhibitory effect against the NCI-N87 cell line and OE-19 cell line than the combination treatment of trastuzumab and pertuzumab.

The above results indicate that the developed humanized antibody hz1E11 of 1E11 has a binding ability comparable to that of existing 1E11 and slightly improved anticancer efficacy.

The anticancer efficacy of the developed humanized antibody hz1E11 of 1E11 by co-administration with trastuzumab was verified in a xenograft model using NCI-N87. Antibodies and trastuzumab isotype controls, hz1E11, trastuzumab, hz1E11, and trastuzumab, respectively, developed in mice with cancer through NCI-N87 transplantation were injected twice a week (intraperitoneal administration). The isotype control antibody and trastuzumab were administered at a dose of 10 mg/kg. In the case of co-administration, hz1E11 and trastuzumab were administered in a 1:1 ratio, and at doses of 1 mg/kg, 2.5 mg/kg, 5 mg/kg, and 10 mg/kg for each antibody. When hz1E11 and trastuzumab were co-administered, it was confirmed that the growth of cancer decreased in a dose-dependent manner (FIG. 9). The anticancer efficacy seen by administering trastuzumab at 10 mg/kg could be seen through the administration of hz1E11 and trastuzumab at 1 mg/kg, respectively. When hz1E11 and trastuzumab were administered in an amount of 5 mg/kg or more, it was confirmed that not only inhibited the growth of cancer, but also reduced the formed cancer.

Example 13: Confirmation of the binding site of the developed antibody

In order to identify a site that is important for antigen binding of the developed antibody, an alanine scanning analysis was performed to confirm the binding ability by converting the amino acid of the region corresponding to the CDR3 of the heavy and light chain to alanine. Histidine (H), leucine (L), glycine (G), glycine (G), threonine (T) and serine corresponding to 95, 96, 97, 98, 99 and 100a according to Kabat numbering among the CDR3 regions of the heavy chain chain (S) and glutamine (Q), glutamine (Q), leucine (L), tyrosine (Y), serine (S) and threonine corresponding to 89, 90, 91, 92, 93 and 94 of the CDR3 regions of the light chain (T) was transformed into alanine using the QuikChange Site-directed Mutagenesis kit (Stratagene, #200518). Among the CDR3s of the heavy chain, A100 was excluded from the analysis because the developed antibody had alanine. After each modified antibody was expressed in bacteria, a periplasmic extract was obtained, the expression was confirmed through dot-blot, and the binding ability of HER2-ECD was analyzed by ELISA method (see Fig. 10).

Mutated sequence Variation position Binding capacity to Her2-ECD (absorbance at 450nm) CDR-L3 A QLYSTPWT Q89A 1.711 Q A LYSTPWT Q90A 1.705 QQ A YSTPWT L91A 1.492 QQL A STPWT Y92A 1.803 QQLY A TPWT S93A 1.733 QQLYS A PWT T94A 1.628 CDR-H3 A LGGTASFDY H95A 1.59 H A GGTASFDY L96A 1.66 HL A GTASFDY G97A 1.08 HLG A TASFDY G98A 0.051 HLGG A ASFDY T99A 0.839 HLGGTA A FDY S100aA 1.597 Parent antibody hz1E11 1.75

As can be seen in Table 6 and FIGS. 10A-10B, the modified antibodies were expressed at a similar level, whereas in the case of G98A of the heavy chain, the binding ability was completely lost, and in the case of G97A, the binding ability was significantly reduced. there was. Mutations in other sites did not significantly affect antigen-antibody binding.

Example 13: Enhancing the affinity of the developed antibody

In order to enhance the affinity of the developed antibody, a library was developed in which CDR3 of the light or heavy chain was randomized. F, D, Y corresponding to F100b, D101, Y102, which are amino acid numbers according to Kabat numbering in the CDR3 of the heavy chain chain, and P, W, T corresponding to P95, W96, and T97 in the CDR3 of the light chain, are each position of a human antibody. It was excluded from randomization because it is an amino acid that is frequently found in. A phage antibody library was developed in which the heavy and light chain CDR3 amino acids except for the amino acids listed in the above technique were randomized to 20 amino acids (Phage display: a laboratory manual, Carlos Barbas III, et al., Cold Spring Harbor Laboratory Press). . The primers used at this time are for the regions corresponding to the CDR3 of the heavy and light chains, and the first and second positions of the codons corresponding to the amino acids to be randomized are adenine (A), cytosine (C), and guanine (G). And thymine (T) were mixed at the same ratio to enter randomly, and the third position was synthesized so that guanine (G) or cytosine (C) at the same ratio was used.

In order to select clones with improved affinity from the developed library, HER2-ECD-His protein was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Thermo Scientific, #21435) and used as an antigen for antibody selection. Was used. The developed phage antibody library and biotin-HER2-ECD-His protein were bound at room temperature for 2 hours, and then phage bound to the antigen was isolated using 50 µl of Dynabeads M-270 Streptavidin (Invitrogen, #653.06). This sorting process was performed 4 times, and among the selected colonies, a colony expressing an antibody binding to HER2-ECD was selected through ELISA using periplasmic extract, and the sequence of the antibody expressed in the selected colonies was analyzed by base analysis. It was confirmed through. The amino acid sequences of the CDR3s of the heavy and light chains of the antibody binding to HER2-ECD are summarized in Tables 7 and 8. No. 1 in each table is the amino acid sequence of the CDR3 of the heavy and light chain chains of hz1E11.

CDRH3 sequence of variants selected in the process of affinity enhancement One HLGGTASFDY 14 HLGGMSSFDY 27 HWGGTASFDY 40 PLGGTASFDY 2 AFGGTASFDY 15 HLGGMTSFDY 28 HYGGTASFDY 41 QLAGTASFDY 3 DLGGTASFDY 16 HLGGSSSFDY 29 MNGGTASFDY 42 SFGGTASFDY 4 FWGGTASFDY 17 HLGGTACFDY 30 NFGGTASFDY 43 SHGGTASFDY 5 HCGGTASFDY 18 HLGGTGAFDY 31 NHGGMASFDY 44 SLGGTASFDY 6 HFGGTASFDY 19 HLGGTGSFDY 32 NHGGTASFDY 45 SMGGTASFDY 7 HHGGTASFDY 20 HLGGTSTFDY 33 NIGGTASFDY 46 SNGGTASFDY 8 HIGGTASFDY 21 HLGGTTSFDY 34 NLGGTASFDY 47 SWGGTASFDY 9 HLCSTASFDY 22 HLGSTASFDY 35 NMGGTASFDY 48 SYGGTASFDY 10 HLCVTASFDY 23 HLYRTASFDY 36 NNGGTASFDY 49 YYGGTASFDY 11 HLGGAASFDY 24 HMGGTASFDY 37 NWGGTASFDY 12 HLGGLPSFDY 25 HRGGTASFDY 38 NYGGAASFDY 13 HLGGMASFDY 26 HVGGTASFDY 39 NYGGTASFDY

CDRL3 sequence of variants selected in the process of affinity enhancement One QQLYSTPWT 41 QQLAYEPWT 81 QQMAFVPWT 121 QQMVRTPWT 2 DQLYGTPWT 42 QQLAYSPWT 82 QQMAFYPWT 122 QQMVRVPWT 3 DQMYSTPWT 43 QQLAYTPWT 83 QQMAGFPWT 123 QQMVSIPWT 4 HQLAFTPWT 44 QQLAYVPWT 84 QQMASVPWT 124 QQMYGTPWT 5 LQHNEFPWT 45 QQLGFAPWT 85 QQMAYGPWT 125 QQMYKTPWT 6 QDMSRTPWT 46 QQLGFIPWT 86 QQMAYSPWT 126 QQMYRTPWT 7 QELSTTPWT 47 QQLGFSPWT 87 QQMAYTPWT 127 QQNAFEPWT 8 QEMMRTPWT 48 QQLGFVPWT 88 QQMDFTPWT 128 QQNAFGPWT 9 QNLAYSPWT 49 QQLGYAPWT 89 QQMEHTPWT 129 QQNAFIPWT 10 QNMYGTPWT 50 QQLGYSPWT 90 QQMFAIPWT 130 QQNAFSPWT 11 QQAAFSPWT 51 QQLHSTPWT 91 QQMFGSPWT 131 QQNAFTPWT 12 QQAAYSPWT 52 QQLKNTPWT 92 QQMFRTPWT 132 QQNAFVPWT 13 QQAAYVPWT 53 QQLMRKPWT 93 QQMFSTPWT 133 QQNAYAPWT 14 QQCTSDPWT 54 QQLRASPWT 94 QQMFSVPWT 134 QQNAYGPWT 15 QQHDVGPWT 55 QQLRNLPWT 95 QQMGFSPWT 135 QQNAYNPWT 16 QQIAFGPWT 56 QQLRNSPWT 96 QQMGYAPWT 136 QQNAYSPWT 17 QQIAFNPWT 57 QQLRNVPWT 97 QQMGYSPWT 137 QQNFIAPWT 18 QQIAFSPWT 58 QQLRSAPWT 98 QQMHIFPWT 138 QQNMIVPWT 19 QQIAFTPWT 59 QQLRSSPWT 99 QQMMAVPWT 139 QQNRISPWT 20 QQIAFVPWT 60 QQLRSVPWT 100 QQMMKSPWT 140 QQNRIWPWT 21 QQIAKTPWT 61 QQLRVIPWT 101 QQMMRTPWT 141 QQNRVIPWT 22 QQIAYSPWT 62 QQLSFTPWT 102 QQMMRVPWT 142 QQNRVVPWT 23 QQIAYTPWT 63 QQLSFVPWT 103 QQMRKIPWT 143 QQNSYSPWT 24 QQIAYVPWT 64 QQLSKTPWT 104 QQMRNVPWT 144 QQNVIVPWT 25 QQIFSVPWT 65 QQLSRAPWT 105 QQMRRVPWT 145 QQNVNVPWT 26 QQIGFSPWT 66 QQLSRSPWT 106 QQMRSTPWT 146 QQNYKLPWT 27 QQIGWTPWT 67 QQLSVTPWT 107 QQMSFSPWT 147 QQSAFVPWT 28 QQIMTLPWT 68 QQLSYAPWT 108 QQMSHSPWT 148 QQSAYAPWT 29 QQIREIPWT 69 QQLSYSPWT 109 QQMSKIPWT 149 QQSAYIPWT 30 QQISFMPWT 70 QQLVRIPWT 110 QQMSRVPWT 150 QQSEACPWT 31 QQISFSPWT 71 QQLVRNPWT 111 QQMSYAPWT 151 QQSFNTPWT 32 QQIYITPWT 72 QQLVRTPWT 112 QQMSYGPWT 152 QQSKTVPWT 33 QQKAYAPWT 73 QQLVRVPWT 113 QQMSYIPWT 153 QQTAFGPWT 34 QQKKGIPWT 74 QQLYSSPWT 114 QQMSYSPWT 154 QQTAFSPWT 35 QQKMGNPWT 75 QQMAFAPWT 115 QQMSYTPWT 155 QQTAYAPWT 36 QQKSVAPWT 76 QQMAFGPWT 116 QQMSYVPWT 156 QQTAYSPWT 37 QQLAFAPWT 77 QQMAFIPWT 117 QQMTRVPWT 157 QQTRRTPWT 38 QQLAFMPWT 78 QQMAFNPWT 118 QQMVIIPWT 158 QQTSFAPWT 39 QQLAFSPWT 79 QQMAFSPWT 119 QQMVREPWT 159 QQVAYSPWT 40 QQLAFVPWT 80 QQMAFTPWT 120 QQMVRSPWT 160 QQVFAIPWT

Among the selected clones, the clones with _{improved K off} were analyzed using Biacore 3000 (GE Healthcare). The HER2-ECD-His protein was immobilized on the CM5 sensor chip by the amine coupling method using ECD/NHS. Antibodies were expressed from each clone using IPTG, and then periplasmic extract was obtained and bound to HER2-ECD-His. The k _off value of each antibody was analyzed using BIAevaluation software. Through this, _{antibodies with improved k off} were selected (see Table 9). Table 9 summarizes representative examples of clones developed by the present inventors, showing improved or similar k _{off values compared to the hz1E11 parent antibody.}

Clone hz1E11 Chain of mutation introduction k _off Degree of reduction
(Fold) LCDR3 HCDR3 hz1E11 QQLYSTPWT HLGGTASFDY - 1.13E-03 1.0 M3 -L-A1-3-1A12
( hz1E11 -133) QQNAYAPWT HLGGTASFDY L 3.72E-05 30.4 M3 -L-A1-3-1F11
( hz1E11 -154) QQTAFSPWT HLGGTASFDY L 1.17E-04 9.7 M1 -L-A1-3-1 C3
( hz1E11 -3) DQMYSTPWT HLGGTASFDY L 1.52E-04 7.4 M3-H-A1-2-1B12 QQLYSTPWT NYGGTASFDY H 2.12E-04 5.3 M1-H-A1-2-1B5 QQLYSTPWT HFGGTASFDY H 4.53E-04 2.5 M3-H-A1-1-1C11 QQLYSTPWT SWGGTASFDY H 5.67E-04 2.0 M3-H-A1-1-1A10 QQLYSTPWT SYGGTASFDY H 1.16E-03 1.0 M3-LH-A1-1-1H1 QQNFIAPWT NYGGTASFDY LH 1.66E-03 0.7 M3-LH-A3-3-2B1 QQLVRNPWT NFGGTASFDY LH 1.65E-04 6.5 M3-L-A3-4-2E8 QQIAYVPWT HLGGTASFDY L 1.80E-04 5.9 M1-LH-A3-3-1A6 QQLVRTPWT NYGGTASFDY LH 1.84E-04 5.8 M3-H-A3-3-2A7 QQLSYSTPWT NFGGTASFDY H 2.26E-04 4.7 M3-LH-A3-3-2F1 QQNAYNPWT HLGGTASFDY L 2.45E-04 4.4 M3-LH-A3-3-2A5 QQMFSTPWT HWGGTASFDY LH 2.98E-04 3.6 M3-H-A3-3-2D8 QQLYSTPWT HWGGTASFDY H 3.30E-04 3.2 M3-LH-A1-2-2F4 QQLVRIPWT NLGGTASFDY LH 3.31E-04 3.2 M3-L-A1-3-2C2 QQLGFIPWT HLGGTASFDY L 5.26E-04 2.0 M3-H-A3-1-2F3 QQLYSTPWT NLGGTASFDY H 7.70E-04 1.4 M3-H-A2-1-1F2 QQLYSTPWT SNGGTASFDY H 1.27E-03 0.8

As can be seen in Table 9, various CDRH3s represented by Formula 1 and CDRL3 represented by Formula 2 herein show similar or improved k _off values to CDRH3 and CDRL3 of the parent antibody hz1E11 antibody.

The experiment was performed afterwards using hz1E11-3, hz1E11-133, and hz1E11-154 among the randomized light chain CDR3 sequences.

The heavy chain variable regions of hz1E11-3, hz1E11-133 and hz1E11-154 are the same as those of hz1Ell, and the amino acid sequences of the light chain variable regions are described in SEQ ID NO: 247, 249 and 251, respectively.

In order to verify the increase in the affinity of the selected three antibodies, antibodies were produced in the form of IgG. Goat anti-human IgG (Invitrogen, #H10500) was fixed to the CM5 sensor chip at 2000 RU by the ECD/NHS method. Thereafter, the antibody was bound at a rate of 50 μl/min for 5 minutes, and a buffer was flowed for 5 minutes to stabilize. The concentration of the antibody used to bind the antibody was 0.4 μg/mL for Trastuzumab (TRA), 0.8 μg/mL for pertuzumab (PER), and 1 μg/mL for hz1E11 and the selected antibodies. After stabilizing the antibody, HER2-ECD-His protein was bound at a concentration of 640 nM, 320 nM, 160 nM, 80 nM, 40 nM, 20 nM, 0 nM at a rate of 50 μl/min for 4 minutes and then for 15 minutes Separated by flowing a buffer. After analysis of each concentration, 10 mM Glycine (pH 1.5) was used to regenerate and the following analysis was performed. The affinity of the antibody was analyzed using the BIAevaluation software. The analysis results are summarized in Table 10.

Antibody k _a
(1/Ms) k _d (1/s) R _max K _D
(M) hz1E11 3.60E+04 8.30E-04 61 2.30E-08 hz1E11-3 3.80E+04 2.00E-04 64 5.20E-09 hz1E11-133 6.40E+04 9.90E-05 68 1.50E-09 hz1E11-154 8.60E+04 9.90E-05 65 1.10E-09 TRA 4.90E+04 1.50E-04 43 3.00E-09 PER 3.80E+04 1.20E-04 56 3.30E-09

In Table 10, k _a , k _d R _max and K _D represent association rate constants, dissociation rate constants, maximum binding capacity and equilibrium dissociation constants, respectively.

As can be seen in Table 10, hz1E11-133 and hz1E11-154 showed a k _off value, that is, k _d value, which is 8.4 times reduced compared to hz1E11, and k _on value, that is, k _a There was a slight increase in the value. As a result, in terms of final affinity, hz1E11-133 was 1.5 nM and hz1E11-154 was 1.1 nM, which was improved 15 times and 20 times compared to hz1E11.

Example 14: Verification of anti-cancer efficacy of antibody with enhanced affinity

The anticancer efficacy of the affinity-enhanced antibody was confirmed in gastric cancer and breast cancer overexpressing HER2. Cancer cell viability according to concentration was analyzed when each antibody was treated alone or in combination with trastuzumab to NCI-N87 and OE-19 cells, which are HER2 overexpressing gastric cancer cell lines, and BT-474, which is HER2 overexpressing breast cancer cells.

As can be seen in Figures 11a-11f, it can be seen that the hz1E11-3, hz1E11-133, and hz1E11-154 antibodies with improved affinity show more improved efficacy in single treatment and parallel treatment compared to hz1E11.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> AbcClon, Inc <120> Antibodies Capable of Binding Specifically to HER2 <130> PP140042 <150> 10-2013-0055912 <151> 2013-05-16 <160> 251 <170> KopatentIn 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 1E11 antibody <400> 1 Ser Tyr Thr Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 1E11 antibody <400> 2 Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 1E11 antibody <400> 3 His Leu Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 1E11 antibody <400> 4 Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala 1 5 10 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 1E11 antibody <400> 5 Ala Thr Ser Leu Ala Asp 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 1E11 antibody <400> 6 Gln Gln Leu Tyr Ser Thr Pro Trp Thr 1 5 <210> 7 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 1E11 heavy chain variable region <400> 7 gaggtgaagt tggtggagtc tgggggaggt ttagtgcagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatacca tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtg ggtcgcatac attagtaatg gtggtggtag cacttactat 180 ccagacactg taaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acggccatgt attactgtgc aagacatcta 300 ggtgggactg cctcttttga ctactggggc caaggcacca cggtcaccgt ctcctca 357 <210> 8 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 1E11 heavy chain variable region <400> 8 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Gly Thr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 9 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 1E11 light chain variable region <400> 9 gacattcaga tgactcagtc tcctgcctcc cagtctgcat ctctgggaga aagtgtcacc 60 atcacatgcc tggcaagtca gaccattggt acatggttag catggtatca gcagaaacca 120 gggaaatctc ctcagctcct gatttatgtt gcaaccagcc tggcagatgg ggtcccatca 180 aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct 240 gaagattttg taagttatta ctgtcaacaa ctttacagta ctccgtggac gttcggtgga 300 gggaccaagc tggagctgaa a 321 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 1E11 light chain variable region <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly 1 5 10 15 Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Val Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 11 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Constant region of human IgG kappa chain <400> 11 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagttcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttaa 324 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Constant region of human IgG kappa chain <400> 12 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 13 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Constant region of human IgG heavy chain <400> 13 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc cccgggtaaa 990 <210> 14 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Constant region of human IgG heavy chain <400> 14 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 15 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> LF-1 <400> 15 ccgatcgata tggagacaga cacactcctg ctatgggtac tgctgctctg ggttccaggt 60 tccacgtggg atattcagat g 81 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> LR-1 <400> 16 cggcgtacgt ttcagctcca gcttgg 26 <210> 17 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> HF-1 <400> 17 ccgatcgata tggagacaga cacactcctg ctatgggtac tgctgctctg ggttccaggt 60 tccacgtggg aggtgaagct 80 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HR-1 <400> 18 cgggctagct gaggagacgg tgac 24 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Ck-F <400> 19 ggagctgaaa cgtacggtgg ctgcacc 27 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Ck-R <400> 20 ccgctcgagt taacactctc ccctgttg 28 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> CH-F <400> 21 caccgtctcc tcagctagca ccaagggccc atcg 34 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CH-R <400> 22 ccgctcgagt catttacccg gggacaggga g 31 <210> 23 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of hz1E11 heavy chain variable region <400> 23 gaagtgcagc tagtggagtc aggcggcggt ttagtgcagc ccgggggctc cctcaggctg 60 tcttgcgccg caagtggatt taccttcagc agctatacaa tgtcttgggt cagacaagcg 120 cctggaaagg gactggagtg ggtagcctac atctccaacg ggggcggaag tacgtattat 180 ccagatactg ttaaagggag atttacaatt agcagagaca atgccaagaa ttccttgtat 240 ctgcagatga actctctcag agctgaagat accgcagtct actattgtgc tagacacctg 300 ggtgggaccg cctccttcga ctactggggc cagggtacac ttgttactgt gtcatct 357 <210> 24 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of hz1E11 heavy chain variable region <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Gly Thr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 25 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of hz1E11 light chain variable region <400> 25 gatatccaga tgacacaaag cccatcatct ttatctgcca gcgtgggaga tagagtgacc 60 atcacatgtc tggcatcaca gaccatcgga acttggttgg cctggtacca gcaaaaacca 120 ggcaaggccc ctaagctgct gatttacgtc gcaacgagtc tcgctgacgg tgtgccttcc 180 agattttccg gttccggcag cggcacagac tttactctga caattagttc cctgcagccc 240 gaggacttcg ctacttatta ctgccagcag ctttatagca ccccctggac cttcgggcag 300 gggaccaaag ttgaaataaa g 321 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of hz1E11 light chain variable region <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRH3 of hz1E11 <400> 27 Ala Leu Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRH3 of hz1E11 <400> 28 His Ala Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRH3 of hz1E11 <400> 29 His Leu Ala Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRH3 of hz1E11 <400> 30 His Leu Gly Ala Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRH3 of hz1E11 <400> 31 His Leu Gly Gly Ala Ala Ser Phe Asp Tyr 1 5 10 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRH3 of hz1E11 <400> 32 His Leu Gly Gly Thr Ala Ala Phe Asp Tyr 1 5 10 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRL3 of hz1E11 <400> 33 Ala Gln Leu Tyr Ser Thr Pro Trp Thr 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRL3 of hz1E11 <400> 34 Gln Ala Leu Tyr Ser Thr Pro Trp Thr 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRL3 of hz1E11 <400> 35 Gln Gln Ala Tyr Ser Thr Pro Trp Thr 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRL3 of hz1E11 <400> 36 Gln Gln Leu Ala Ser Thr Pro Trp Thr 1 5 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRL3 of hz1E11 <400> 37 Gln Gln Leu Tyr Ala Thr Pro Trp Thr 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Alanine substituted sequence of CDRL3 of hz1E11 <400> 38 Gln Gln Leu Tyr Ser Ala Pro Trp Thr 1 5 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 39 Ala Phe Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 40 Asp Leu Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 41 Phe Trp Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 42 His Cys Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 43 His Phe Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 44 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 44 His His Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 45 His Ile Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 46 His Leu Cys Ser Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 47 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 47 His Leu Cys Val Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 48 His Leu Gly Gly Ala Ala Ser Phe Asp Tyr 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 49 His Leu Gly Gly Leu Pro Ser Phe Asp Tyr 1 5 10 <210> 50 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 50 His Leu Gly Gly Met Ala Ser Phe Asp Tyr 1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 51 His Leu Gly Gly Met Ser Ser Phe Asp Tyr 1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 52 His Leu Gly Gly Met Thr Ser Phe Asp Tyr 1 5 10 <210> 53 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 53 His Leu Gly Gly Ser Ser Ser Phe Asp Tyr 1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 54 His Leu Gly Gly Thr Ala Cys Phe Asp Tyr 1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 55 His Leu Gly Gly Thr Gly Ala Phe Asp Tyr 1 5 10 <210> 56 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 56 His Leu Gly Gly Thr Gly Ser Phe Asp Tyr 1 5 10 <210> 57 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 57 His Leu Gly Gly Thr Ser Thr Phe Asp Tyr 1 5 10 <210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 58 His Leu Gly Gly Thr Thr Ser Phe Asp Tyr 1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 59 His Leu Gly Ser Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 60 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 60 His Leu Tyr Arg Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 61 His Met Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 62 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 62 His Arg Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 63 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 63 His Val Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 64 His Trp Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 65 His Tyr Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 66 Met Asn Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 67 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 67 Asn Phe Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 68 Asn His Gly Gly Met Ala Ser Phe Asp Tyr 1 5 10 <210> 69 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 69 Asn His Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 70 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 70 Asn Ile Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 71 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 71 Asn Leu Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 72 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 72 Asn Met Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 73 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 73 Asn Asn Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 74 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 74 Asn Trp Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 75 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 75 Asn Tyr Gly Gly Ala Ala Ser Phe Asp Tyr 1 5 10 <210> 76 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 76 Asn Tyr Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 77 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 77 Pro Leu Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 78 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 78 Gln Leu Ala Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 79 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 79 Ser Phe Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 80 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 80 Ser His Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 81 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 81 Ser Leu Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 82 Ser Met Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 83 Ser Asn Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 84 Ser Trp Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 85 Ser Tyr Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRH3 of hz1E11 <400> 86 Tyr Tyr Gly Gly Thr Ala Ser Phe Asp Tyr 1 5 10 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 87 Asp Gln Leu Tyr Gly Thr Pro Trp Thr 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 88 Asp Gln Met Tyr Ser Thr Pro Trp Thr 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 89 His Gln Leu Ala Phe Thr Pro Trp Thr 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 90 Leu Gln His Asn Glu Phe Pro Trp Thr 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 91 Gln Asp Met Ser Arg Thr Pro Trp Thr 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 92 Gln Glu Leu Ser Thr Thr Pro Trp Thr 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 93 Gln Glu Met Met Arg Thr Pro Trp Thr 1 5 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 94 Gln Asn Leu Ala Tyr Ser Pro Trp Thr 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 95 Gln Asn Met Tyr Gly Thr Pro Trp Thr 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 96 Gln Gln Ala Ala Phe Ser Pro Trp Thr 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 97 Gln Gln Ala Ala Tyr Ser Pro Trp Thr 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 98 Gln Gln Ala Ala Tyr Val Pro Trp Thr 1 5 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 99 Gln Gln Cys Thr Ser Asp Pro Trp Thr 1 5 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 100 Gln Gln His Asp Val Gly Pro Trp Thr 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 101 Gln Gln Ile Ala Phe Gly Pro Trp Thr 1 5 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 102 Gln Gln Ile Ala Phe Asn Pro Trp Thr 1 5 <210> 103 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 103 Gln Gln Ile Ala Phe Ser Pro Trp Thr 1 5 <210> 104 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 104 Gln Gln Ile Ala Phe Thr Pro Trp Thr 1 5 <210> 105 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 105 Gln Gln Ile Ala Phe Val Pro Trp Thr 1 5 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 106 Gln Gln Ile Ala Lys Thr Pro Trp Thr 1 5 <210> 107 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 107 Gln Gln Ile Ala Tyr Ser Pro Trp Thr 1 5 <210> 108 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 108 Gln Gln Ile Ala Tyr Thr Pro Trp Thr 1 5 <210> 109 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 109 Gln Gln Ile Ala Tyr Val Pro Trp Thr 1 5 <210> 110 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 110 Gln Gln Ile Phe Ser Val Pro Trp Thr 1 5 <210> 111 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 111 Gln Gln Ile Gly Phe Ser Pro Trp Thr 1 5 <210> 112 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 112 Gln Gln Ile Gly Trp Thr Pro Trp Thr 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 113 Gln Gln Ile Met Thr Leu Pro Trp Thr 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 114 Gln Gln Ile Arg Glu Ile Pro Trp Thr 1 5 <210> 115 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 115 Gln Gln Ile Ser Phe Met Pro Trp Thr 1 5 <210> 116 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 116 Gln Gln Ile Ser Phe Ser Pro Trp Thr 1 5 <210> 117 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 117 Gln Gln Ile Tyr Ile Thr Pro Trp Thr 1 5 <210> 118 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 118 Gln Gln Lys Ala Tyr Ala Pro Trp Thr 1 5 <210> 119 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 119 Gln Gln Lys Lys Gly Ile Pro Trp Thr 1 5 <210> 120 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 120 Gln Gln Lys Met Gly Asn Pro Trp Thr 1 5 <210> 121 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 121 Gln Gln Lys Ser Val Ala Pro Trp Thr 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 122 Gln Gln Leu Ala Phe Ala Pro Trp Thr 1 5 <210> 123 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 123 Gln Gln Leu Ala Phe Met Pro Trp Thr 1 5 <210> 124 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 124 Gln Gln Leu Ala Phe Ser Pro Trp Thr 1 5 <210> 125 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 125 Gln Gln Leu Ala Phe Val Pro Trp Thr 1 5 <210> 126 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 126 Gln Gln Leu Ala Tyr Glu Pro Trp Thr 1 5 <210> 127 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 127 Gln Gln Leu Ala Tyr Ser Pro Trp Thr 1 5 <210> 128 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 128 Gln Gln Leu Ala Tyr Thr Pro Trp Thr 1 5 <210> 129 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 129 Gln Gln Leu Ala Tyr Val Pro Trp Thr 1 5 <210> 130 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 130 Gln Gln Leu Gly Phe Ala Pro Trp Thr 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 131 Gln Gln Leu Gly Phe Ile Pro Trp Thr 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 132 Gln Gln Leu Gly Phe Ser Pro Trp Thr 1 5 <210> 133 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 133 Gln Gln Leu Gly Phe Val Pro Trp Thr 1 5 <210> 134 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 134 Gln Gln Leu Gly Tyr Ala Pro Trp Thr 1 5 <210> 135 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 135 Gln Gln Leu Gly Tyr Ser Pro Trp Thr 1 5 <210> 136 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 136 Gln Gln Leu His Ser Thr Pro Trp Thr 1 5 <210> 137 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 137 Gln Gln Leu Lys Asn Thr Pro Trp Thr 1 5 <210> 138 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 138 Gln Gln Leu Met Arg Lys Pro Trp Thr 1 5 <210> 139 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 139 Gln Gln Leu Arg Ala Ser Pro Trp Thr 1 5 <210> 140 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 140 Gln Gln Leu Arg Asn Leu Pro Trp Thr 1 5 <210> 141 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 141 Gln Gln Leu Arg Asn Ser Pro Trp Thr 1 5 <210> 142 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 142 Gln Gln Leu Arg Asn Val Pro Trp Thr 1 5 <210> 143 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 143 Gln Gln Leu Arg Ser Ala Pro Trp Thr 1 5 <210> 144 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 144 Gln Gln Leu Arg Ser Ser Pro Trp Thr 1 5 <210> 145 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 145 Gln Gln Leu Arg Ser Val Pro Trp Thr 1 5 <210> 146 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 146 Gln Gln Leu Arg Val Ile Pro Trp Thr 1 5 <210> 147 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 147 Gln Gln Leu Ser Phe Thr Pro Trp Thr 1 5 <210> 148 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 148 Gln Gln Leu Ser Phe Val Pro Trp Thr 1 5 <210> 149 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 149 Gln Gln Leu Ser Lys Thr Pro Trp Thr 1 5 <210> 150 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 150 Gln Gln Leu Ser Arg Ala Pro Trp Thr 1 5 <210> 151 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 151 Gln Gln Leu Ser Arg Ser Pro Trp Thr 1 5 <210> 152 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 152 Gln Gln Leu Ser Val Thr Pro Trp Thr 1 5 <210> 153 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 153 Gln Gln Leu Ser Tyr Ala Pro Trp Thr 1 5 <210> 154 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 154 Gln Gln Leu Ser Tyr Ser Pro Trp Thr 1 5 <210> 155 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 155 Gln Gln Leu Val Arg Ile Pro Trp Thr 1 5 <210> 156 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 156 Gln Gln Leu Val Arg Asn Pro Trp Thr 1 5 <210> 157 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 157 Gln Gln Leu Val Arg Thr Pro Trp Thr 1 5 <210> 158 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 158 Gln Gln Leu Val Arg Val Pro Trp Thr 1 5 <210> 159 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 159 Gln Gln Leu Tyr Ser Ser Pro Trp Thr 1 5 <210> 160 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 160 Gln Gln Met Ala Phe Ala Pro Trp Thr 1 5 <210> 161 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 161 Gln Gln Met Ala Phe Gly Pro Trp Thr 1 5 <210> 162 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 162 Gln Gln Met Ala Phe Ile Pro Trp Thr 1 5 <210> 163 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 163 Gln Gln Met Ala Phe Asn Pro Trp Thr 1 5 <210> 164 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 164 Gln Gln Met Ala Phe Ser Pro Trp Thr 1 5 <210> 165 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 165 Gln Gln Met Ala Phe Thr Pro Trp Thr 1 5 <210> 166 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 166 Gln Gln Met Ala Phe Val Pro Trp Thr 1 5 <210> 167 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 167 Gln Gln Met Ala Phe Tyr Pro Trp Thr 1 5 <210> 168 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 168 Gln Gln Met Ala Gly Phe Pro Trp Thr 1 5 <210> 169 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 169 Gln Gln Met Ala Ser Val Pro Trp Thr 1 5 <210> 170 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 170 Gln Gln Met Ala Tyr Gly Pro Trp Thr 1 5 <210> 171 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 171 Gln Gln Met Ala Tyr Ser Pro Trp Thr 1 5 <210> 172 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 172 Gln Gln Met Ala Tyr Thr Pro Trp Thr 1 5 <210> 173 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 173 Gln Gln Met Asp Phe Thr Pro Trp Thr 1 5 <210> 174 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 174 Gln Gln Met Glu His Thr Pro Trp Thr 1 5 <210> 175 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 175 Gln Gln Met Phe Ala Ile Pro Trp Thr 1 5 <210> 176 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 176 Gln Gln Met Phe Gly Ser Pro Trp Thr 1 5 <210> 177 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 177 Gln Gln Met Phe Arg Thr Pro Trp Thr 1 5 <210> 178 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 178 Gln Gln Met Phe Ser Thr Pro Trp Thr 1 5 <210> 179 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 179 Gln Gln Met Phe Ser Val Pro Trp Thr 1 5 <210> 180 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 180 Gln Gln Met Gly Phe Ser Pro Trp Thr 1 5 <210> 181 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 181 Gln Gln Met Gly Tyr Ala Pro Trp Thr 1 5 <210> 182 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 182 Gln Gln Met Gly Tyr Ser Pro Trp Thr 1 5 <210> 183 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 183 Gln Gln Met His Ile Phe Pro Trp Thr 1 5 <210> 184 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 184 Gln Gln Met Met Ala Val Pro Trp Thr 1 5 <210> 185 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 185 Gln Gln Met Met Lys Ser Pro Trp Thr 1 5 <210> 186 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 186 Gln Gln Met Met Arg Thr Pro Trp Thr 1 5 <210> 187 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 187 Gln Gln Met Met Arg Val Pro Trp Thr 1 5 <210> 188 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 188 Gln Gln Met Arg Lys Ile Pro Trp Thr 1 5 <210> 189 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 189 Gln Gln Met Arg Asn Val Pro Trp Thr 1 5 <210> 190 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 190 Gln Gln Met Arg Arg Val Pro Trp Thr 1 5 <210> 191 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 191 Gln Gln Met Arg Ser Thr Pro Trp Thr 1 5 <210> 192 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 192 Gln Gln Met Ser Phe Ser Pro Trp Thr 1 5 <210> 193 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 193 Gln Gln Met Ser His Ser Pro Trp Thr 1 5 <210> 194 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 194 Gln Gln Met Ser Lys Ile Pro Trp Thr 1 5 <210> 195 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 195 Gln Gln Met Ser Arg Val Pro Trp Thr 1 5 <210> 196 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 196 Gln Gln Met Ser Tyr Ala Pro Trp Thr 1 5 <210> 197 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 197 Gln Gln Met Ser Tyr Gly Pro Trp Thr 1 5 <210> 198 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 198 Gln Gln Met Ser Tyr Ile Pro Trp Thr 1 5 <210> 199 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 199 Gln Gln Met Ser Tyr Ser Pro Trp Thr 1 5 <210> 200 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 200 Gln Gln Met Ser Tyr Thr Pro Trp Thr 1 5 <210> 201 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 201 Gln Gln Met Ser Tyr Val Pro Trp Thr 1 5 <210> 202 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 202 Gln Gln Met Thr Arg Val Pro Trp Thr 1 5 <210> 203 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 203 Gln Gln Met Val Ile Ile Pro Trp Thr 1 5 <210> 204 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 204 Gln Gln Met Val Arg Glu Pro Trp Thr 1 5 <210> 205 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 205 Gln Gln Met Val Arg Ser Pro Trp Thr 1 5 <210> 206 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 206 Gln Gln Met Val Arg Thr Pro Trp Thr 1 5 <210> 207 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 207 Gln Gln Met Val Arg Val Pro Trp Thr 1 5 <210> 208 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 208 Gln Gln Met Val Ser Ile Pro Trp Thr 1 5 <210> 209 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 209 Gln Gln Met Tyr Gly Thr Pro Trp Thr 1 5 <210> 210 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 210 Gln Gln Met Tyr Lys Thr Pro Trp Thr 1 5 <210> 211 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 211 Gln Gln Met Tyr Arg Thr Pro Trp Thr 1 5 <210> 212 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 212 Gln Gln Asn Ala Phe Glu Pro Trp Thr 1 5 <210> 213 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 213 Gln Gln Asn Ala Phe Gly Pro Trp Thr 1 5 <210> 214 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 214 Gln Gln Asn Ala Phe Ile Pro Trp Thr 1 5 <210> 215 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 215 Gln Gln Asn Ala Phe Ser Pro Trp Thr 1 5 <210> 216 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 216 Gln Gln Asn Ala Phe Thr Pro Trp Thr 1 5 <210> 217 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 217 Gln Gln Asn Ala Phe Val Pro Trp Thr 1 5 <210> 218 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 218 Gln Gln Asn Ala Tyr Ala Pro Trp Thr 1 5 <210> 219 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 219 Gln Gln Asn Ala Tyr Gly Pro Trp Thr 1 5 <210> 220 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 220 Gln Gln Asn Ala Tyr Asn Pro Trp Thr 1 5 <210> 221 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 221 Gln Gln Asn Ala Tyr Ser Pro Trp Thr 1 5 <210> 222 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 222 Gln Gln Asn Phe Ile Ala Pro Trp Thr 1 5 <210> 223 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 223 Gln Gln Asn Met Ile Val Pro Trp Thr 1 5 <210> 224 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 224 Gln Gln Asn Arg Ile Ser Pro Trp Thr 1 5 <210> 225 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 225 Gln Gln Asn Arg Ile Trp Pro Trp Thr 1 5 <210> 226 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 226 Gln Gln Asn Arg Val Ile Pro Trp Thr 1 5 <210> 227 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 227 Gln Gln Asn Arg Val Val Pro Trp Thr 1 5 <210> 228 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 228 Gln Gln Asn Ser Tyr Ser Pro Trp Thr 1 5 <210> 229 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 229 Gln Gln Asn Val Ile Val Pro Trp Thr 1 5 <210> 230 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 230 Gln Gln Asn Val Asn Val Pro Trp Thr 1 5 <210> 231 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 231 Gln Gln Asn Tyr Lys Leu Pro Trp Thr 1 5 <210> 232 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 232 Gln Gln Ser Ala Phe Val Pro Trp Thr 1 5 <210> 233 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 233 Gln Gln Ser Ala Tyr Ala Pro Trp Thr 1 5 <210> 234 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 234 Gln Gln Ser Ala Tyr Ile Pro Trp Thr 1 5 <210> 235 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 235 Gln Gln Ser Glu Ala Cys Pro Trp Thr 1 5 <210> 236 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 236 Gln Gln Ser Phe Asn Thr Pro Trp Thr 1 5 <210> 237 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 237 Gln Gln Ser Lys Thr Val Pro Trp Thr 1 5 <210> 238 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 238 Gln Gln Thr Ala Phe Gly Pro Trp Thr 1 5 <210> 239 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 239 Gln Gln Thr Ala Phe Ser Pro Trp Thr 1 5 <210> 240 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 240 Gln Gln Thr Ala Tyr Ala Pro Trp Thr 1 5 <210> 241 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 241 Gln Gln Thr Ala Tyr Ser Pro Trp Thr 1 5 <210> 242 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 242 Gln Gln Thr Arg Arg Thr Pro Trp Thr 1 5 <210> 243 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 243 Gln Gln Thr Ser Phe Ala Pro Trp Thr 1 5 <210> 244 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 244 Gln Gln Val Ala Tyr Ser Pro Trp Thr 1 5 <210> 245 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Randomized sequence of CDRL3 of hz1E11 <400> 245 Gln Gln Val Phe Ala Ile Pro Trp Thr 1 5 <210> 246 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Nucleoide sequence of hz1E11-3 light chain variable region <400> 246 gatatccaga tgacacaaag cccatcatct ttatctgcca gcgtgggaga tagagtgacc 60 atcacatgtc tggcatcaca gaccatcgga acttggttgg cctggtacca gcaaaaacca 120 ggcaaggccc ctaagctgct gatttacgtc gcaacgagtc tcgctgacgg tgtgccttcc 180 agattttccg gttccggcag cggcacagac tttactctga caattagttc cctgcagccc 240 gaggacttcg ctacttatta ctgcgaccag atgtacagca cgccctggac cttcgggcag 300 gggaccaaag ttgaaataaa g 321 <210> 247 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of hz1E11-3 light chain variable region <400> 247 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Asp Gln Met Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 248 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Nucleoide sequence of hz1E11-133 light chain variable region <400> 248 gatatccaga tgacacaaag cccatcatct ttatctgcca gcgtgggaga tagagtgacc 60 atcacatgtc tggcatcaca gaccatcgga acttggttgg cctggtacca gcaaaaacca 120 ggcaaggccc ctaagctgct gatttacgtc gcaacgagtc tcgctgacgg tgtgccttcc 180 agattttccg gttccggcag cggcacagac tttactctga caattagttc cctgcagccc 240 gaggacttcg ctacttatta ctgccagcag aatgcttatg cgccctggac cttcgggcag 300 gggaccaaag ttgaaataaa g 321 <210> 249 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of hz1E11-133 light chain variable region <400> 249 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Tyr Ala Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 250 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Nucleoide sequence of hz1E11-154 light chain variable region <400> 250 gatatccaga tgacacaaag cccatcatct ttatctgcca gcgtgggaga tagagtgacc 60 atcacatgtc tggcatcaca gaccatcgga acttggttgg cctggtacca gcaaaaacca 120 ggcaaggccc ctaagctgct gatttacgtc gcaacgagtc tcgctgacgg tgtgccttcc 180 agattttccg gttccggcag cggcacagac tttactctga caattagttc cctgcagccc 240 gaggacttcg ctacttatta ctgccagcag acggcttttt ctccctggac cttcgggcag 300 gggaccaaag ttgaaataaa g 321 <210> 251 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of hz1E11-154 light chain variable region <400> 251 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Ala Phe Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

Claims (21)

(a) a heavy chain variable region comprising CDRHl of SEQ ID No. 1, CDRH2 of SEQ ID No. 2 and CDRH3 of SEQ ID No. 43, and CDRL1 of SEQ ID No. 4, CDRL2 of SEQ ID No. 5, An antibody or antigen-binding fragment thereof for human epidermal growth factor receptor 2 (HER2) comprising a light chain variable region comprising CDRL3 of SEQ ID NO: 6;
(b) a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 84, and CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: An antibody or antigen-binding fragment thereof for human epidermal growth factor receptor 2 (HER2) comprising a light chain variable region comprising CDRL3 of SEQ ID NO: 6;
(c) a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 64, and CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: An antibody or antigen-binding fragment thereof for human epidermal growth factor receptor 2 (HER2) comprising a light chain variable region comprising CDRL3 of SEQ ID NO: 178;
(d) a heavy chain variable region comprising CDRHl of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 64, and CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: An antibody or antigen-binding fragment thereof for human epidermal growth factor receptor 2 (HER2) comprising a light chain variable region comprising CDRL3 of SEQ ID NO: 6;
(e) a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 71, and CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, An antibody to HER2 (Human Epidermal Growth Factor Receptor 2) or an antigen-binding fragment thereof comprising a light chain variable region comprising CDRL3 of Sequence Listing 155;
(f) a heavy chain variable region comprising CDRH1 of Sequence Listing 1, CDRH2 of Sequence Listing 2 and CDRH3 of Sequence Listing 3, and CDRL1 of Sequence Listing 4, CDRL2 of Sequence Listing 5, and An antibody or antigen-binding fragment thereof for human epidermal growth factor receptor 2 (HER2) comprising a light chain variable region comprising CDRL3 of SEQ ID NO: 131; or
(g) a heavy chain variable region comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 71, and CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: An antibody or antigen-binding fragment thereof to a human epidermal growth factor receptor (HER2) comprising a light chain variable region comprising CDRL3 of SEQ ID NO: 6.
delete delete delete delete delete delete delete delete delete delete delete delete delete delete (a) a pharmaceutically effective amount of the antibody or its antigen-binding fragment of HER2 of claim 1; And (b) a pharmaceutically acceptable carrier.
18. The composition of claim 16, wherein the pharmaceutical composition further comprises a Trastuzumab antibody.
The method of claim 16, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, stomach cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, Skin cancer, thyroid cancer, parathyroid cancer or urethral cancer.
delete delete A cancer diagnostic kit comprising an antibody against HER2 of claim 1 or an antigen-binding fragment thereof.

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