KR101515535B1 - Anti-ErbB2 Antibody Variants - Google Patents

Abstract

The present invention relates to a composition for the prophylaxis or treatment of cancer comprising an anti-ErbB2 antibody mutant or an antigen-binding fragment thereof, a nucleic acid molecule encoding the same, and an antibody variant or an antigen-binding fragment thereof. The antibody variant of the present invention has an improved affinity for ErbB2 and exhibits excellent cancer cell proliferation inhibitory activity as compared to 4D5, a conventional commercially available antibody therapy.

Description

Anti-ErbB2 Antibody Variants {Anti-ErbB2 Antibody Variants}

The present invention relates to a composition for the prevention or treatment of cancer comprising an anti-ErbB2 antibody variant or antigen-binding fragment thereof, a nucleic acid molecule encoding the same, and the antibody variant or a lower binding fragment thereof.

HER2 (ErbB2) is a type of receptor tyrosin kinase such as EGFR (ErbB1), HER3 (ErbB3) and HER4 (ErbB4), and is present in the cell membrane and plays an important role in cell growth, differentiation and survival. It is known (Jaclyn et al. Clin Breast Cancer . (2008) 8:38-49). Unlike other HER family proteins, HER2 is not activated depending on the ligand and always acts in an activated state. Normally, about 20,000 cells are expressed on the cell membrane in normal cells, but about 20,000,000 cells, which are 100 times that of cancer cells, are overexpressed. It is known (Shepard et al. J Clin Immunol. (1991) 11:117-127). This overexpression of HER2 excessively induces not only the HER2-HER2 homodimer, but also the heterodimer with HER1 or HER3, which is another HER family, and as a result, it causes the proliferation and growth of the cells to be transformed into cancer cells. Promote (Mayumi et al. (2006) Clin Cancer Res . 12:7242-7251).

Until now, breast cancer (25-30%), ovarian cancer (15-30%), gastric cancer (23%), lung cancer (11-32%), kidney cancer (30-40%), rectal cancer (17-90%), Overexpression of HER2 has been reported in various cancer cells such as pancreatic cancer (26-45%), bladder cancer (44%), prostate cancer (12%) and head and neck cancer (29-39%) ( Cancer Immunol Immunother (2004) 53:166-175; Clin Cancer Res (2006) 12:4377s-4383s; Br J Cancer (2004) 91:1195-1199; Cancer (2002) 94:2584-2589; Cancer (2003) 98: 66-73; Int J Oncol (2005) 27: 681-685; Int J Pancreatol (1995) 17:15-21; Int J Cancer (2000) 87:349-359; Ann Oncol (2001) 12:S15-S19; J Pathol (2004) 204:317-325). In addition, overexpression of HER2 was observed in endometrial cancer, salivary gland cancer, colon cancer and thyroid cancer (King et al., Science (1985) 229:974; Yokota et al., Lancet : (1986) 1:765-767; Fukushigege) et al., Mol Cell Biol . (1986) 6:955-958; Guerin et al., Oncogene Res . (1988) 3:21-31; Cohen et al., Oncogene (1989) 4:81-88; Yonemura et al., Cancer Res . (1991) 51:1034; Borst et al., Gynecol . Oncol , (1990) 38:364; Weiner et al., Cancer Res . (1990) 50:421-425; Kern et al., Cancer Res . (1990) 50:5184; Park et al., Cancer Res . (1989) 49:6605; Zhau et al., Mol . Carcinog . (1990) 3:254-257; Aasland et al. Br . J. Cancer (1988) 57:358-363; Williams et al. Pathobiology (1991) 59:46-52; McCann et al., Cancer (1990) 65:88-92).

The only anticancer treatment targeting HER2 is Herceptin (Herceptin ^ⓡ , Trastuzumab, 4D5) developed by Genentech and sold since 1998. Its indication is that HER2 is overexpressed in metastatic breast cancer or early breast cancer. ( J. Clin . Oncol . (1999) 17:2639-2264). Herceptin is known to inhibit cancer cell survival and proliferation by inhibiting homodimer production between HER2-HER2 by acting on the extracellular domain IV of HER2 ( Ann Oncol (2007) 18:977-984).

However, despite the success of Herceptin as a target anticancer drug for HER2, it exhibits a response effect of 12-34% when administered alone, 38-50% when administered in combination, and 2-7% when administered alone, 11-28 when administered in combination. Up to% of patients have reported abnormal heart disease, and about 1 in 10 women cannot receive Herceptin medication due to concerns about an increased risk of heart disease ( N Engl J Med (2007) 357:39-51). Therefore, there is a demand for the development of a subsequent antibody with fewer side effects than Herceptin, and Omnitarg developed by Genetech is currently in phase 3 clinical trials ( Clin Cancer Res (2006) 12:4436s-4440s). However, Omnitarg has an advantage over Herceptin in terms of side effects, but has a disadvantage in that its efficacy is inferior. For this reason, there is an urgent need for novel HER2 antibodies or improved forms thereof, for example 4D5 antibody variants with increased affinity or potency.

On the other hand, antibodies can bind to antigens by forming an antibody-specific structure with six complementarity-determining regions (CDRs) that form a loop and a framework region (FR) extending from the CDRs. Yes (Fell et al. (1989) Immunol Ser . 43:181-202). FR has a conserved sequence compared to the CDR, but the CDR loop consists of a wide variety of sequences for each antibody (Wu et al. (1970) J Expt Med . 132:211-250). Such antibody sequence diversity is _{achieved by gene rearrangement of VH, D H} , J _H (heavy chain sequence) or V _κ , J _κ or V _λ and J _λ (light chain sequence) in B cells. In addition to gene rearrangement, high somatic cell mutations occur through addition of nucleic acids or point mutations, so that antibodies with antigen specificity and high affinity can be produced in the body.

In general, obtaining a high affinity antibody for an antigen is not easy because the surface of the antigen to be exposed is limited. In particular, this phenomenon occurs better than when performing in vitro selection from a naive phage display library. Therefore, the affinity of antibodies generally obtained from naive phage display library is only 10-100 nM level (Iwai et al. (2010) Protein Eng Des Sel . 23:185-193).

Strategies for improving the affinity of an antibody can be divided into a random approach and a targeted approach (Sheedy et al. (2007) Biotechnol Adv . 5(4):333-52). Targeted mutagenesis is a strategy of mutating specific CDRs or FRs, that is, specific amino acids. Targeted PCR, CDR walking, site-directed mutagenesis, and CDR targeting A method such as a hotspot (CDR target hotspot) can be used. Random mutagenesis is a strategy for mutating variable domains, and methods such as error-prone PCR, DNA shuffling, and chain shuffling can be used (Kim et al. (2006) Adv). Drug Deliv Rev. 58:657-667). Various variants made in this way are produced as a library, and various variant libraries are displayed on the surface of the phage to be screened. In addition, yeast displays and ribosomal displays are also used (Rader et al. (1997) Curr Opin Biotechnol . 8:503-508; Zahnd et al. (2004) J Biol Chem . 279(18):18870-18877).

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 an antibody variant with improved antigen affinity and/or cancer cell proliferation inhibitory activity than 4D5, a humanized antibody against ErbB2. Accordingly, the present inventors produced an antibody library with a random mutagenesis strategy using targeting mutagenesis targeting CDRs and error-prone PCR, and selected anti-ErbB2 antibody variants using phage display technology, and selected antibody variants The present invention was completed by confirming that not only shows high antigen affinity compared to the parent antibody, but also can significantly inhibit the proliferation of cancer cells.

Accordingly, an object of the present invention is to provide an anti-ErbB2 antibody variant or antigen-binding fragment thereof.

Another object of the present invention is to provide a nucleic acid molecule encoding the heavy chain variable domain of the antibody variant or antigen-binding fragment thereof.

Another object of the present invention is to provide a nucleic acid molecule encoding the light chain variable domain of the antibody variant or antigen-binding fragment thereof.

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

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides (a) a light chain variable domain; And (b) a heavy chain variable domain having two or more amino acid substitutions selected from the group consisting of the following amino acid substitutions, or an antigen-binding fragment thereof:

In the amino acid sequence of SEQ ID NO: 1, Pro at position 41 according to the Kabat numbering system is substituted with Arg, Gly at position 96 is substituted with Asn, Gly at position 97 is substituted with Ala, and position 98 Asp at position 98 is substituted with Trp, Asp at position 98 is substituted with Lys, Ala at position 100b is substituted with Ser, Met at position 100c is substituted with Phe, Asp at position 101 is substituted with Ala, Asp at position 101 is substituted with Val Substitution, Tyr at position 102 is substituted with His, and Tyr at position 102 is substituted with Leu.

The present inventors have tried to develop an antibody variant with improved antigen affinity and/or cancer cell proliferation inhibitory activity than 4D5, a humanized antibody against ErbB2. Accordingly, the present inventors produced an antibody library with a random mutagenesis strategy using targeting mutagenesis targeting CDRs and error-prone PCR, and selected anti-ErbB2 antibody variants using phage display technology, and selected antibody variants It was confirmed that not only shows high antigen affinity compared to the parent antibody, but also can significantly inhibit the proliferation of cancer cells.

Hereinafter, it will be described in detail below.

Ⅰ. term- ErbB2 Antibody Variant And antigen-binding fragments thereof

The antibody variant of the present invention has a specific binding ability to ErbB2.

As used herein, the term "antibody variant" refers to an amino acid substitution variant including a variable domain in which at least two amino acids are substituted in each of the heavy chain variable domain and/or the light chain variable domain of the humanized antibody 4D5 against ErbB2. it means. That is, the present invention provides an antibody variant with improved antigen affinity and cancer cell proliferation inhibitory activity by substituting an amino acid at a specific position in the heavy and/or light chain variable domains of the parent antibody 4D5.

As confirmed in the following examples, the antibody variant of the present invention has improved affinity for ErbB2 by up to 8 times compared to 4D5, which is the parent antibody (Table 5), and also promotes proliferation of gastric cancer cells with about 3.5 times superior activity compared to 4D5. Inhibit (Fig. 8).

The heavy chain variable domain of the 4D5 antibody includes the amino acid sequence of SEQ ID NO: 1, and the light chain variable domain of the 4D5 antibody includes the amino acid sequence of SEQ ID NO: 2.

Antibody variants of the present invention include complete antibody forms as well as antigen binding fragments (antibody fragments) of antibody molecules.

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 has subclasses of 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.

The antigen-binding fragment or antibody fragment of an antibody molecule 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. Fab' 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 generated by disulfide bonds between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Recombination techniques for generating Fv fragments include 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 heavy chain variable region and the single chain variable region 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 entire antibody with papain yields Fab, and digestion with pepsin yields F(ab') ₂ fragments), preferably It can be produced through genetic recombination technology.

In the present invention, the antibody is preferably in the form of Fv 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 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of kappa or lambda type.

As used herein, the term "heavy chain" includes a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _{H3 comprising an amino acid sequence having a sufficient variable region sequence to confer antigen specificity.} Means both full-length heavy chains and fragments thereof. In addition, the term "light chain" as used herein refers to 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 It means all short stories.

According to a preferred embodiment of the present invention, the heavy chain variable domain comprises the following amino acid substitutions:

(I) Asp at position 98 according to the Kabat numbering system in the amino acid sequence of SEQ ID NO: 1 is substituted with Trp, Met at position 100c is substituted with Phe, Asp at position 101 is substituted with Ala, and Tyr at position 102 Substitute this Leu (SEQ ID NO: 3);

(Ii) Gly at position 96 according to the Kabat numbering system in the amino acid sequence of SEQ ID NO: 1 is substituted with Asn, Gly at position 97 is substituted with Ala, Asp at position 98 is substituted with Lys, and Ala at position 100b Ser substituted, Met at position 100c is substituted with Phe, Asp at position 101 is substituted with Val, and Tyr at position 102 is substituted with His (SEQ ID NO: 4); or

(Iii) Pro at position 41 according to the Kabat numbering system in the amino acid sequence of SEQ ID NO: 1 is substituted with Arg, Asp at position 98 is substituted with Trp, Met at position 100c is substituted with Phe, Asp at position 101 This Ala was substituted, and Tyr at position 102 was substituted with Leu (SEQ ID NO: 6).

Position 41 according to the Kabat numbering system, mentioned while describing the amino acid substitution of the heavy chain variable domain in the present specification, refers to Pro, the 41st amino acid of SEQ ID NO: 1, and position 96 is of SEQ ID NO: 1. It refers to the 100th amino acid Gly, position 97 refers to the 101st amino acid of SEQ ID NO: 1, Gly, position 98 refers to Asp, the 102nd amino acid of SEQ ID NO: 1, and position 100b is the sequence listing It means Ala, which is the 106th amino acid of SEQ ID NO: 1, position 100c means Met, which is the 107th amino acid of SEQ ID NO: 1, and position 101 means Asp, which is the 108th amino acid of SEQ ID NO: 1, and position 102 refers to Tyr, which is the 109th amino acid of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 2, or comprises the following amino acid substitutions:

In the amino acid sequence of SEQ ID NO: 2, Ser at position 50 according to the Kabat numbering system is substituted with Thr, Ala at position 51 is substituted with Thr, Ser at position 52 is substituted with Thr, and Phe at position 53 is substituted with Trp. Substitution, Leu at position 54 is substituted with Pro, Thr at position 72 is substituted with Ser, His at position 91 is substituted with Tyr, Thr at position 93 is substituted with Gln, Thr at position 93 is substituted with Asn, Two or more amino acid substitutions selected from the group consisting of substitution of Pro for Ala, substitution for Pro at position 96 with Val, and substitution for Thr at position 97 with Ser.

According to a more preferred embodiment of the present invention, the light chain variable domain comprises the following amino acid substitutions:

(I) In the amino acid sequence of SEQ ID NO: 2 in the sequence listing, Thr at position 93 according to the Kabat numbering system is substituted with Gln, Pro at position 96 is substituted with Ala, and Thr at position 97 is substituted with Ser (SEQ ID NO: 5 order); or

(Ii) Ser at position 50 according to the Kabat numbering system in the amino acid sequence of SEQ ID NO: 2 is substituted with Thr, Ala at position 51 is substituted with Thr, Ser at position 52 is substituted with Thr, and Phe at position 53 Substitute this Trp, Leu at position 54 is substituted with Pro, Thr at position 72 is substituted with Ser, His at position 91 is substituted with Tyr, Thr at position 93 is substituted with Asn, and Pro at position 96 is substituted with Val ( SEQ ID NO: 7).

Position 50 according to the Kabat numbering system mentioned while describing the amino acid substitution of the light chain variable domain in the present specification refers to Ser, the 50th amino acid of SEQ ID NO: 2, and position 51 is of SEQ ID NO: 2 It refers to the 51st amino acid Ala, position 52 refers to Ser, the 52nd amino acid of SEQ ID NO: 2, position 53 refers to Phe, the 53rd amino acid of SEQ ID NO: 2, and position 54 refers to the sequence listing. It means Leu, the 54th amino acid of SEQ ID NO: 72, position 72 refers to Thr, the 72nd amino acid of SEQ ID NO: 2, and position 91 refers to His, the 91st amino acid of SEQ ID NO: 2, and position 93 refers to Thr, the 93rd amino acid of SEQ ID NO: 2, position 96 refers to Pro, the 96th amino acid of SEQ ID NO: 2, and position 97 refers to Thr, the 97th amino acid of SEQ ID NO: 2 it means.

According to a preferred embodiment of the present invention, the antibody variant or antigen-binding fragment thereof of the present invention comprises: (a) a heavy chain variable domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6; And (b) a light chain variable domain selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 7.

According to a more preferred embodiment of the present invention, the antibody variant or antigen-binding fragment thereof of the present invention comprises the following light chain variable domain and heavy chain variable domain:

(I) the light chain variable domain of SEQ ID NO: 2 and the heavy chain variable domain of SEQ ID NO: 3;

(Ii) a light chain variable domain of SEQ ID NO: 2 and a heavy chain variable domain of SEQ ID NO: 4;

(Iii) the light chain variable domain of SEQ ID NO: 5 and the heavy chain variable domain of SEQ ID NO: 3; or

(Iv) the light chain variable domain of SEQ ID NO: 7 and the heavy chain variable domain of SEQ ID NO: 6.

Antibodies of the present invention include 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.

According to a preferred embodiment of the present invention, the antibody of the present invention is a humanized antibody.

The antibody variant or antibody fragment of the present invention may include not only the anti-ErbB2 antibody variant sequence of the present invention described herein, but also biological equivalents thereof, within a range that can specifically recognize HER2. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. 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, substitutions are 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).

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, Thr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and 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. (1981) 2:482 ; Needleman and Wunsch, J. Mol . Bio . (1970) 48:443; Pearson and Lipman, Methods in Mol . Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al., Nuc. Acids Res . (1988) 16:10881-90; Huang et al., Comp . Appl . BioSci . (1992) 8:155-65 and Pearson et al., Meth . Mol . Biol . (1994) 24:307-31. NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol. (1990) 215:403-10) can be accessed from NBCI, etc., and on the Internet, such as blastp, blasm, blastx, tblastn and tblastx. It can be used in conjunction with a sequence analysis program. The BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Meanwhile, the antibody variant or antibody fragment of the present invention may be an immunoconjugate combined with a functional molecule. Since HER2 is a molecule expressed on the surface of cancer cells, it can be used for the prevention, treatment and diagnosis of cancer that overexpresses HER2 by additionally binding a functional molecule to the antibody of the present invention. The functional molecules include chemicals, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents and enzyme inhibitors.

Preferred functional molecules for coupling with the antibody variants or fragments thereof of the present invention are chemicals, cytokines or chemokines, more preferably chemicals. The chemical substance is an anticancer agent, for example, acibicin, aclarubicin, acodazole, acronycin, adozelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutetimide, Amonapide, Ampligen, Amsacrine, Androgens, Anguidine, Apidicholine Glycinate, Asarei, Asparaginase, 5-Azacytidine, Azathioprine, Bacillus Calmette-Guerine (BCG ), Bakers Antipol, Beta-2-dioxythioguanosine, Bisantrene HCl, Bleomycin Sulfate, Bulseophan, Butionine Sulfoximine, BWA773U82, BW 502U83/HCl, BW 7U85 Mesylate, Cerasemide , Carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroid, Corynebacterium parvum, CPT-11, Crisnatol, Cyclocytidine, Cyclophosphamide, Cytarabine, Cytembena, Dabis Maleate, Decarbazine, Dactinomycin, Daunorubicin HCl, Diazauridine, Dexrazoic Acid, Deion Hydrogalactitol, diaziquone, dibromodulcitol, didemin B, diethyldithiocarbamate, diclycoaldehyde, dihydro-5-azacytine, doxorubicin, ethinomycin, dedatrexate, Edelfosine, Eplolnithine, Ellios Solution, Elsamitrusin, Epirubicin, Esorubicin, Estramustine Phosphate, Estrogen, Ethanidazole, Ethiophos, Etoposide, Padrazole, Pazarabine, Penreti Nide, filgrastim, finasteride, flavone acetic acid, phloxuridine, fludarabine phosphate, 5'-fluorouracil, Fluosol™, flutamide, gallium nitrate, gemcytabine, gosererin acetate, hefsulfame, Hexamethylene bisacetamide, homoharingtonin, hydrazine sulfate, 4-hydroxyandrostenedione, hydrodiurea, idarubicin HCl, ifosfamide, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium , Leuprolide acetate, levamisol, liposome daunorubicin, liposome capture doxorubicin , Romustine, Ronidamine, Mytansine, Mechloretamine Hydrochloride, Melphalan, Menogaryl, Merbaron, 6-mercaptopurine, Mesna, Bacillus Calete-Guerine Methanol Extract, Methotrexate, N-Methyl Formamide, mifepristone, mitoguazone, mitomycin-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, Streptonimustine, Streptozosine, Sulopener, Suramine Sodium, Tamoxifen, Taxorere, Tegapur, Teniposide, Terephthalamidine, Teroxirone, Thioguanine, Thiotepa, Thymidine Injection, Thiazofurine, Topotecan, Toremifen, Tretinoin, Trifluoroperazine Hydrochloride, Trifluridine, Trimetrexate, TNF (tumor necrosis factor), uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidin, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, Melparan, Taxotel, Taxol, and mixtures thereof, but are not limited thereto.

Ⅱ. Nucleic Acid Molecules and Recombinant Vectors

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the heavy chain variable domain of the antibody variant or antigen-binding fragment thereof of the present invention.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the light chain variable domain of the antibody variant or antigen-binding fragment thereof of the present invention.

As used herein, 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. Also includes modified analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the heavy and light chain variable domains of the present invention can be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable domain is a nucleic acid molecule encoding a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6. , The nucleic acid molecule encoding the light chain variable domain is a nucleic acid molecule encoding a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 7.

According to a preferred embodiment of the present invention, the nucleic acid molecule of the present invention may be in a form contained within a nucleic acid molecule encoding the entire heavy chain region or a nucleic acid molecule encoding the entire light chain region.

The nucleic acid molecule of the present invention is interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The actual 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) a nucleic acid molecule encoding the heavy chain variable domain of the present invention; And (b) a nucleic acid molecule encoding the light chain variable domain of the present invention.

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, and the like, preferably a plasmid vector.

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

As used herein, 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, and thus Thus, the control sequence regulates the transcription and/or translation of the other nucleic acid sequence.

According to a preferred embodiment of the present invention, the recombinant vector of the present invention comprises (a) a nucleic acid molecule encoding a heavy chain variable domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6; And (b) a nucleic acid molecule encoding a light chain variable domain selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 7.

According to a more preferred embodiment of the present invention, the recombinant vector of the present invention comprises the following nucleic acid molecules:

(I) a nucleic acid molecule encoding the heavy chain variable domain of SEQ ID NO: 3 and a nucleic acid molecule encoding the light chain variable domain of SEQ ID NO: 2;

(Ii) a nucleic acid molecule encoding the heavy chain variable domain of SEQ ID NO: 4 and a nucleic acid molecule encoding the light chain variable domain of SEQ ID NO: 2;

(Iii) a nucleic acid molecule encoding the heavy chain variable domain of SEQ ID NO: 3 and a nucleic acid molecule encoding the light chain variable domain of SEQ ID NO: 5; or

(Iv) A nucleic acid molecule encoding the heavy chain variable domain of SEQ ID NO: 6 and a nucleic acid molecule encoding the light chain variable domain of SEQ ID NO: 7.

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 prokaryotic cell is used as a host, a strong promoter capable of progressing transcription (eg, tac promoter, lac Promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding site for initiation of translation, and It is common to include a transcription/translation termination sequence. E. coli as a host cell (e.g., HB101, BL21, DH5α, etc.) are used, E. coli Promoter and operator sites of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , (1984) 158:1018-1024) and the left-facing promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., .. Ann Rev Genet, (1980 ) 14:. 399-445) may be used as a control region. When Bacillus bacteria is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringensis ( Appl . Environ . Microbiol . (1998)) 64:3932-3938; Mol . Gen. Genet . (1996) 250:734-741) or any promoter that can be expressed in Bacillus bacteria can be used as a regulatory site.

On the other hand, the recombinant vector of the present invention is a plasmid often used in the art (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phages (eg, λgt4·λB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg, SV40, etc.) can be manufactured.

According to a preferred embodiment of the present invention, the recombinant vector of the present invention can be produced by engineering a pCL expression vector, preferably a pCLS05 (Korean Application No. 10-2011-0056685) expression vector.

On the other hand, 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 a 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 (CMV) promoter, HSV tk Promoter, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epsteinbar virus (EBV) and promoter of Loews sacoma virus (RSV)) can be used, and transcription termination sequence It generally has a polyadenylation sequence as. Preferably, the recombinant vector of the present invention comprises a CMV promoter.

The recombinant vector of the present invention may be fused with other sequences 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 recombinant 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, neomycin And resistance genes for tetracycline.

The vector expressing the antibody variant of the present invention may be a vector system in which the light and heavy chains are simultaneously expressed in one vector, or a system in which the light and heavy chains are expressed in separate vectors, respectively. In the latter case, both vectors are introduced into host cells through co-transfomation and targeted transformation. Simultaneous transformation is a method of simultaneously introducing each vector DNA encoding a light chain and a heavy chain into a host cell, and then selecting cells expressing both the light and heavy chains. Target transformation involves selecting cells transformed with a vector containing a light chain (or heavy chain) and transforming the selected cells expressing a light chain with a vector containing a heavy chain (or light chain) to express both light and heavy chains. This is a method of finally selecting cells. In the following examples, an antibody was prepared using a vector system in which the light chain and the heavy chain are simultaneously expressed in one vector.

Ⅲ. Transformant

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

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, Escherichia coli (Escherichia coli), Bacillus sp, Streptomyces, such as Bacillus subtilis and Bacillus Chuo ringen sheath (Streptomyces), Pseudomonas (Pseudomonas) (e.g., Pseudomonas footage is (Pseudomonas putida )), Proteus mirabilis ( Proteus mirabilis ) or Staphylococcus (for example, Staphylococcus carnosus )).

Suitable eukaryotic host cells of the vector are the genus Aspergillus (Aspergillus species), such as Pichia pastoris (Pichia pastoris ), Saccharomyces cerevisiae ), Schizosaccharomyces and Neurospora crassa , other lower eukaryotic cells, cells of higher eukaryotes such as insect-derived cells, and from plants or mammals One cell can be used. Preferably, the host cells are monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK). Cells, MDCK, myeloma cell lines, HuT 78 cells or 293 cells, more preferably Chinese hamster ovary cells.

When using microorganisms such as E. coli , the productivity is higher than that of animal cells, but it is not desirable for the production of intact Ig antibodies due to glycosylation problems, but it can be used for production of Fab and Fv. have.

In the present specification, "transformation" and/or "transfection" into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and as known in the art, a standard suitable for the host cell You can do it by choosing a technique. These methods include electroporation, protoplasm fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fibers, agrobacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and drying/inhibition mediated transformation methods, and the like are included, but are not limited thereto.

IV. term- ErbB2 Antibody Variant Or a method for producing an antigen-binding fragment thereof

According to another aspect of the present invention, the present invention comprises the steps of (a) culturing a host cell transformed with the recombinant vector of the present invention; And (b) expressing the anti-ErbB2 antibody variant or antigen-binding fragment thereof in the host cell.

In the production of the antibody, the transformed host cell may be cultured according to an appropriate medium and culture conditions known in the art. Such cultivation process can be easily adjusted and used by those skilled in the art according to the selected strain. These various culture methods are described in various literatures (e.g., James M. Lee, Biochemical Engineering , Prentice-Hall International Editions, 138-176). Cell culture is divided into suspension culture and adhesion culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method. The medium used for cultivation must adequately satisfy the requirements of the specific strain.

In animal cell culture, the medium contains various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, Alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL) and soybean meal, inorganic such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. It contains a nitrogen source. These nitrogen sources may be used alone or in combination. The medium may contain, as personnel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt. In addition, it may contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

The pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture in an appropriate manner during the culture. In addition, during cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C.

The antibody obtained by culturing the transformed host cell may be used without purification, and further purified with high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography, may be used. Among them, the method using chromatography is most commonly used, and the type and order of the column can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc. depending on the characteristics of the antibody and the culture method.

Ⅴ. Pharmaceutical composition for preventing or treating cancer

According to another aspect of the present invention, the present invention provides (a) a pharmaceutically effective amount of the anti-ErbB2 antibody variant or antigen-binding fragment thereof of the present invention; And (b) it provides a pharmaceutical composition for the prevention or treatment of cancer comprising a pharmaceutically acceptable carrier.

The antibody prepared by the above method binds with high affinity to ErbB2 (HER2) and can inhibit the growth of cancer that overexpresses HER2, so it can be used for the prevention and treatment of cancer alone or with a conventional pharmaceutically acceptable carrier. Do.

As used herein, the term "prevention" refers to any action that inhibits or delays the progression of cancer by administration of the composition of the present invention, and "treatment" refers to inhibition of the development of cancer, alleviation of cancer, or elimination of cancer. Means.

According to a preferred embodiment of the present invention, cancer, which is a disease applied to the composition of the present invention, is a cancer that overexpresses HER2, more preferably breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, rectal cancer, pancreatic cancer, bladder cancer, prostate cancer. Cancer, head and neck cancer, endometrial cancer, salivary gland cancer or thyroid cancer, more preferably breast cancer or gastric cancer, and most preferably gastric cancer.

As used herein, the term "cancer overexpressing HER2" is a cancer that has a significantly higher level of HER2 on the surface of cancer cells compared to non-cancerous cells of the same tissue type. This overexpression can occur either by gene amplification or by increased transcription or translation.

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 described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration It can be administered by the like. When administered orally, since the protein or peptide is digested, the oral composition should be coated with an active agent or formulated to protect it from degradation in the stomach. In addition, the pharmaceutical composition can be administered by any device capable of moving the active substance to the target cell.

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 0.001-100 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 skilled in the art. Or it can be prepared by incorporating it into 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 composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

The antibody variant can be injected into the body in the form of an antibody variant-therapeutic agent (functional molecule) conjugate and used for the treatment of cancer, and the description thereof is as described above. Several suitable and preferred conditions for targeting a drug to a specific target site are described, for example, in Trouet et al., Plenum. Press , New York and London, (1982) 19-30.

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

(a) The antibody variant of the present invention binds to ErbB2 with high affinity. In particular, the antibody variant of the present invention has improved affinity for ErbB2 by up to 8 times compared to 4D5, a conventional commercial antibody therapeutic agent.

(b) The antibody variant of the present invention exhibits excellent cancer cell proliferation inhibitory activity. In particular, the anti-proliferation activity of the antibody variant of the present invention is about 3.5 times better than that of 4D5.

(c) Therefore, the antibody variant of the present invention can effectively prevent or treat cancer even with a small amount.

1 shows a part of a vector expressing a separate transcript for display of scFv. The open reading frame encodes the OmpA secretion signal sequence, light chain variable domain, linker sequence (L), heavy chain variable domain, c-myc tag sequence and viral envelope protein.
2 is a graph showing the results of a monoclonal ELISA for selecting E. coli clones that produce scFv-pIII molecules that bind to ErbB2.
3 is a heavy chain variable domain of humanized antibody 4D5 (SEQ ID NO: 1), heavy chain variable domains of AH06 and A058 (SEQ ID NO: 3), heavy chain variable domain of AH16 (SEQ ID NO: 4), and heavy chain variable of A091 The amino acid sequences of the domains (SEQ ID NO: 6) are compared and shown.
Figure 4 is the amino acid sequence of the light chain variable domain of humanized antibodies 4D5, AH06, AH16 (SEQ ID NO: 2), light chain variable domain of A058 (SEQ ID NO: 5) and light chain variable domain of A091 (SEQ ID NO: 7) Is shown in comparison.
5 shows SDS-PAGE results of anti-ErbB2 antibody variants according to the present invention produced and purified in animal cells.
6 to 8 show the results of analysis of the cell proliferation inhibitory activity of anti-ErbB2 antibody variants. Figure 6: D98W; Figure 7: A058, A091; Figure 8: AH06, AH16. Purified IgG was evaluated in NCI-N87 cells for 6 days.

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. Various CDR Residue Having phage-displayed scFv Library creation

A phage-displayed scFv library was constructed using a phagemid vector that generates a display in a form in which scFv and pIII are conjugated. The schematic structure of the vector is shown in FIG. 1. The vector contains the variable domain of the antibody under the control of the IPTG-inducible Plac promoter, and the linker sequence is GGGGSGGGSGGSS (SEQ ID NO: 8). A method for producing an anti-ErbB2 antibody (4D5) as a parent antibody and a variable domain sequence are known in U.S. Patent Nos. 5,821,337 and 6,054,297.

The library was generated using the only "stop template" version of the scFv display vector. In this example, a template pCMTG phagemid vector in which the TGA stop codon was inserted into Kabat residue 48 of the light chain was used (IG Therapy Co., Korea). No stop codon was introduced into the heavy chain CDR3. A mutagenesis oligonucleotide having an NNK codon at the position to be diversified was used to introduce CDR diversity and simultaneously remove the stop codon. This generated an open reading frame encoding scFv library members fused to homodimerized c-myc and pIII.

The heavy chain CDR3 and light chain CDR3 regions were preferentially changed. In order to change the heavy chain CDR3 region, PCR was performed using HF (SEQ ID NO: 19), LN01_R primer (SEQ ID NO: 9), HF (SEQ ID NO: 19) and LN02_R primer (SEQ ID NO: 10). Using a C1000 Thermal cycler device (Bio-Rad, USA), the amplification process was performed with Ex taq (Takara, Japan) according to the manufacturer's instructions as follows: denaturation at 95°C for 20 seconds, denaturation at 57°C for 30 seconds. 27 cycles of primer binding and amplification process extending at 72°C for 45 seconds were performed. And, PCR was performed as follows using a pair of LN03_F (SEQ ID NO: 11), LR primer (SEQ ID NO: 17), LN04_F (SEQ ID NO: 12) and LR primers to change the light chain CDR3 region: 95°C for 20 seconds During denaturation, primer binding at 57°C for 30 seconds, and amplification process extending at 72°C for 45 seconds performed 27 cycles.

Two PCR procedures were performed to construct a library that randomly induces mutations using the NNK codon as a template for the antibodies selected from the heavy chain CDR3 and light chain CDR3 variants as a template. PCR was performed 27 cycles of denatured at 95°C for 20 seconds, primer binding at 57°C for 30 seconds, and extended at 72°C for 45 seconds. LF (SEQ ID NO: 18) was used as the forward primer, and the A fragment was obtained using a primer in which LN05_R (SEQ ID NO: 13) and LN06_R (SEQ ID NO: 14) were mixed in a ratio of 1:1 as the reverse primer, and LN0506_F (SEQ ID NO: No. 15) and HR primer (SEQ ID No. 20) were used to obtain a B fragment. PCR was performed using a pair of LF (SEQ ID NO: 18) and HR primer (SEQ ID NO: 20) as templates for A fragment and B fragment. In addition, a library was additionally constructed using LF (SEQ ID NO: 18) and LN07_R primer (SEQ ID NO: 16) as templates for antibodies selected from heavy chain CDR3 and two light chain (CDRL2, CDRL3) variants.

Using the heavy chain CDR3, light chain CDR3, and light chain CDR2 mutant antibodies selected from the library as a template, error-prone PCR was performed to randomly induce mutations targeting both the CDRs and the framework portions to obtain an antibody library. In order to make, PCR process was performed twice as follows: The first PCR was denatured at 95°C for 20 seconds using GeneMorpII Random mutagenesis kit (Stratagene, USA), and primer binding at 57°C for 30 seconds and The amplification process extending for 45 seconds at 72° C. was performed 32 cycles, and the second PCR was denatured at 95° C. for 20 seconds using Ex taq (Takara, Japan), primer binding at 58° C. for 30 seconds, and 72° C. The amplification process extending for 45 seconds at 20 cycles was performed; First, PCR was performed on a fragment containing a light chain using LF (SEQ ID NO: 18) and LR (SEQ ID NO: 17) and a fragment containing a heavy chain using a pair of HF (SEQ ID NO: 19) and HR primers (SEQ ID NO: 20). Each was amplified through. The two fragments of the light and heavy chain were PCR with a pair of LF (SEQ ID NO: 18) and HR primer (SEQ ID NO: 20) to obtain scFv-type fragments to prepare a library. The sequences of the primers used in PCR are shown in Table 1.

Primer name order Sequence number LN01_R ACTAGTGCTACTCACGGTCACCAGAGTTCCCTGTCCCCAGTAATCCATGGCGTAMNNGCCMNNMNNMNNMNNTCTAGAGCAGTAGTAC Sequence 9 LN02_R ACTAGTGCTACTCACGGTCACCAGAGTTCCCTGTCCCCAMNNMNNMNNMNNGTAGAAGCCATCACC Sequence 10 LN03_F GACTTCGCTACGTACTACTGCNNKNNKNNKNNKACCACTCCTCCGAC Sequence 11 LN04_F GACTTCGCTACGTACTACTGCCAACAGCACTACNNKACTNNKNNKNNKTTCGGACAAGGCAC Sequence 12 LN05_R TGAATCTAGATGGCACACCMNNMNNMNNGAAMNNMNNMNNGTAGATCAGCAGCTTC Sequence 13 LN06_R TGAATCTAGATGGCACACCMNNMNNMNNCCAMNNMNNMNNGTAGATCAGCAGCTTC SEQ ID NO: 14 LN0506_F GGTGTGCCATCTAGATTCAGTG Sequence 15 LN07_R ACTAGTGCTACTCACGGTCACCAGAGTTCCCTGTCCCCAGTAATCCATMNNGTAMNNGCCMNNMNNMNNMNNTCTAGAGCAGTAGTAC Sequence 16 LR GCGCGCTACTCACGGTC SEQ ID NO: 17 LF GGCCCAGGCGGCCGATATCCAGATGAC Sequence 18 HF GAGCTCATGGATATCCAGATGACCCAGAG SEQ ID NO: 19 HR GCGCGCTACTCACGGTC Sequence 20

The parent antibody contains the amino acid sequence of the variable domain of the humanized antibody 4D5 (the heavy chain variable domain of SEQ ID NO: 1; the light chain variable domain of SEQ ID NO: 2) (Table 2).

Amino acid sequence Sequence list Heavy chain variable domain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS First sequence Light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Second sequence

Libraries LN01 and LN02 were constructed with randomized residues in one heavy chain CDR (CDRH3), and libraries LN03 and LN04 were constructed with randomized residues in one light chain CDR (CDRL3). In addition, libraries LN05, LN06 and LN07 were constructed with randomized residues in two light chain CDRs (CDRL2, CDRL3) and heavy chain CDR3. Specific randomized residues are shown in Table 3. CDR positions were numbered according to Kabat nomenclature. In addition, the variant library including the framework portion as well as the CDR produced through error-prone PCR was named LN08.

The library was electroporated in E. coli XLl-blue (200158, Stratagene, USA) (Sidhu et al. (2000) Methods Enzymol . 328:333-363), transformed cells were grown overnight in the presence of Ex12 helper phage (IG therapy, Korea) to encapsulate phagemid DNA and produce phage particles displaying scFv fragments on the surface thereof. Each library is 5 x 10 ⁸ pcs. Contains more than one member.

Example 2. From the library ErbB2 Selection of specific antibodies

ErbB2 specific antibodies were selected from the antibody library of Example 1. NUNC 96-well Maxisorp immunoplates were coated overnight with a capture target (2 μg/ml) at 4° C. and blocked with Superblock Tris buffered saline (Pierce) for 2 hours. After growing at 37° C. overnight, the phage was concentrated by precipitation with PEG/NaCl, and resuspended in Superblock TBS, 0.05% Tween 20 (Sigma). Thereafter, a phage solution (about 10 ^l2 phage/ml) was added to the coated immunoplate. After incubation for 2 hours for phage binding, the plate was washed 10 times with PBS and 0.05% Tween 20. The bound phage was eluted with 0.1 M glycine (pH 2.2) for 10 minutes, and the eluent was neutralized with 1 M Tris/Cl (pH 9.0). The eluted phage was amplified in E. coli XLl-Blue.

_{K D} of the parent antibody Since the value is at the picomolar level and the affinity with the antigen is very high, it is difficult to select an antibody with improved affinity. Therefore, the present inventors largely used three methods to select an antibody having a higher affinity than the parent antibody. As the first method, the washing time was treated for up to 44 hours to select antibodies with improved off-rate (Chen et al. (1999) J Mol Biol . 293:865-81). As a second method, a pre-elution process was performed using 0.1 M glycine (pH 2.2), followed by a final elution method (Bruin et al. (1999) Nat Biotechnol. 17( 4):397-9), as a third method, a method of removing weakly bound antibodies by treatment with ammonium thiocyanate before elution was used (Macdonald et al. (1988) J Immunol). Methods 106:191-4; Wang et al. (2000) J Immunol Methods . 241(1-2):171-84; Hur et al. (2010) Immunol Lett . 134(1):55-61).

Individual clones selected through the above experiment were grown in a 96-well format in 500 µl of 2YT broth supplemented with carbenicillin and ampicillin, and the culture supernatant was directly used for phage ELISA to bind to the plate coated with the target protein, but BSA A phage-displayed scFv that did not bind to the plate coated with was detected (FIG. 2). Compared to the BSA-coated plate, a clone having an OD value of 0.5 or more on the target protein-coated plate and maintaining avidity even when treated with 1.0 M ammonium thiocyanate was defined as a specific conjugate (variant) for ErbB2. After 4-5 replicates, individual clones were screened to select conjugates for the target protein ErbB2 from the library.

The DNA sequence of the specific conjugate was analyzed. As a result of the analysis, it was confirmed that amino acids were substituted at positions 98, 100c, 101 and 102 of the heavy chain variable domain of the parent antibody 4D5 according to the numbering system according to Kabat for one specific conjugate (AH06). As a result of confirming the sequence of a conjugate other than AH06, Kabat residues 41, 96, 97 or 100b in the heavy chain variable domain in addition to Kabat residues 98, 100c, 101 and 102, Kabat residues 50, 51, 52 in the light chain variable domain , 53, 54, 72, 91, 93, 96 or 97 was confirmed that the amino acid was substituted.

In order to find the optimal specific conjugate, each of the substituted residues was combined and finally selected specific conjugates were named AH06, AH16, A058, and A091. The heavy chain of humanized antibody AH06 is a variant of the humanized 4D5 antibody heavy chain, and contains the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3. Humanized antibody AH16 comprises the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 4. Humanized antibody A058 comprises the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5. Humanized antibody A091 comprises the amino acid sequence of SEQ ID NO: 6 and SEQ ID NO: 7.

The amino acid sequences of the heavy and light chain variable domains of the anti-agent are shown in Table 4. In addition, the amino acid sequences of the parent antibody 4D5 and the heavy and light chain variable domains of the antibody were compared and shown in FIGS. 3 and 4.

Heavy and light chain variable domain amino acid sequences Amino acid sequence Sequence list Heavy chain variable domain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGWGFYAFALWGQGTLVTVSS 3rd sequence Heavy chain variable domain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWNAKGFYSFVHWGQGTLVTVSS Sequence 4 Light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYQTPASFGQGTKVEIK Sequence 5 Heavy chain variable domain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQARGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGWGFYAFALWGQGTLVTVSS Sequence 6 Light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYTTTWPYSGVPSRFSGSRSGTDFSLTISSLQPEDFATYYCQQYYNTPVTFGQGTKVEIK Sequence 7

Example 3. Anti- ErbB2 Of antibodies Light chain And Heavy chain Preparation of whole gene expression vector

The pCLS05 expression vector (Korean Application No. 2011-0056685) was used to prepare the variable domain gene of the scFv-type humanized antibody prepared in Example 2 as the entire gene of the humanized antibody. First, in order to insert the heavy chain variable domain gene of the antibody into the expression vector pCLS05, the heavy chain variable domain gene was cut by reacting with the restriction enzyme EcoRI/XhoI (NEB, USA) for 2 hours at 37° C., and then 1.5% agarose gel Through electrophoresis and ethidium bromide staining, a gene fragment of about 1.4 kb was identified. Likewise, the expression vector pCLS05 was also digested with the restriction enzyme EcoRI/XhoI to confirm the digested expression vector of about 7.5 kb. Thereafter, each gene fragment was recovered using a Qiagen extraction kit (Qiagen, USA), and then reacted overnight at 16° C. using T4 DNA ligase (DNA ligase, Takara Holdings, Japan), and then E. coli DH5α Was transformed to obtain a transformant. The resulting transformant was cultured in LB medium containing 100 µg/ml of ampicillin overnight, and then the plasmid was recovered, and then sequence analysis confirmed that the entire heavy chain gene was inserted into the vector.

In order to insert the entire light chain gene into the pCLS05 expression vector containing the entire heavy chain gene of the humanized antibody, the entire light chain gene was digested by reacting with the restriction enzyme HindIII/BamHI (NEB, USA) for 2 hours at 37°C, followed by 1.5% Through agarose gel electrophoresis and ethidium bromide staining, a gene fragment of about 0.7 kp was identified. Likewise, the expression vector pCLS05 was also digested with the restriction enzyme EcoRI/XhoI to confirm the digested expression vector of about 8.8 Kb. Thereafter, each gene fragment was recovered using a Qiagen extraction kit (Qiagen. USA), and then reacted overnight at 16°C using a T4 DNA ligase (Takara Holdings, Japan), and then E. coli DH5α was transformed. To obtain a transformant. The obtained transformant was cultured in LB medium containing 100 µg/ml of ampicillin overnight, and then the plasmid was recovered, and then it was confirmed that the entire light chain gene was inserted into the vector through sequencing.

Example 4. For the expression of humanized antibodies CHO Cell transformation

In order to measure the activity of humanized antibodies in animal cells, FreeStyle ^TM CHO-s cells (#R800-07, Invitrogen, USA) were transformed using the expression vectors each prepared in Example 3 above. First, in order to cultivate CHO cells, a culture medium in which 8 mM L-glutamine (25030-081, Gibco, USA) was added ^{to FreeStyle TM CHO Expression medium (Invitrogen, USA) was used.} 5% after the carbon dioxide is supplied to culture the cells for 2 to 3 days at 37 ℃ incubator (humidified CO ₂ incubator) which, the cells were recovered and separated for 5 minutes centrifugation at 1,200 rpm at room temperature (25 ℃). Next, the recovered CHO-s cells were stained with a 0.4% trypan blue (Fluka, USA) solution, and the number of cells was measured using a hematocytometer.

CHO-s cells were subcultured with a new medium so that the cell concentration was 5 x 10 ^{5 cells/ml and viability> 95%.} After reacting 132 μg of expression vector and 130 μl FreeStyle ^TM MAX Reagent (#16447-100, Invitrogen, USA) for 10 minutes, mix with the prepared cells and CO ₂ incubator (37° C., 5% CO ₂ , 110 rpm Orbital shaker). ).

Example 5. Purification of humanized antibodies

The transformant obtained in Example 4 was cultured as follows, and then the antibody was purified to obtain a humanized antibody. ^{Specifically, 5 x 10 7} cells were cultured in a 500 ml Erlenmeyer flask in 100 ml of FreeStyle ^™ CHO Expression medium in 5% CO ₂ , 37° C. humidified incubator for 5 days. The cell culture solution was recovered and centrifuged, and then filtered to obtain only the culture solution containing the antibody. In addition, 1 ml of Mabselect (17-5199-01, GE Healthcare, UK), a resin having a binding capacity of 30 mg human IgG/ml, was used in a disposable chromatography column (732-1010, BIO-) to purify only antibodies from the culture medium. RAD, USA). In order to bind the antibody with Protein-A, the cell culture solution expressing the antibody was passed through a Mabselect column, and then passed through a 0.1 M sodium citrate buffer to elute the humanized antibody bound to Protein-A. Thereafter, Tris-Cl (pH 9.0) buffer was added in 1/10 volume of the antibody recovery solution to neutralize the pH of the antibody eluate.

The purified antibody was subjected to buffer change with PBS (pH 7.4). After wetting with MWCO 30000 VIVASPIN (Sartorius, Cat. No. VS2021) with PBS, a purified antibody sample was added and centrifuged at 3,000 rpm, 4° C. for 30 minutes. To the sample concentrated to 0.5 ml, PBS was added in a volume of 10 times so as to be 5 ml, and centrifugation was repeated 3 times to allow buffer exchange more than 1,000 times.

Proteins of the purified antibody were confirmed using NuPAGE Novex Bis-Tris Mini Gels 4-12% gradient (Invitrogen, Cat. No. NP0321). As a result, as shown in FIG. 5, both the heavy chain protein band (50,000 Dalton molecular weight position) and the light chain protein band (25,000 Dalton molecular weight position) of the purified antibody could be confirmed (FIG. 5 ).

Example 6. Anti- ErbB2 Of antibodies ErbB2 Affinity measurement for

The affinity of the anti-ErbB2 antibody variants according to the present invention to ErbB2 was measured. First, Chen et al. (1999) J Mol Biol . 293(4):865-81) to obtain BIAcore data. The binding affinity of the antibody variants for ErbB2 was calculated from the binding and dissociation rate constants measured using the BIAcore™-2000 surface plasmon resonance system (BIAcore, Inc.). The antigen was immobilized on the surface of the M5 sensor chip using the amino coupling method ( Analyt . Biochem . (1991) 198:268-277). ErbB2 was covalently coupled by activating the biosensor chip using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). ErbB2 was buffered to 10 mM sodium acetate, pH 4.8, and diluted to about 30 μg/ml. An aliquot of ErbB2 was injected at a flow rate of 5 μl/min to achieve approximately 250-300 reaction units (RU) of coupled protein. A 1 M ethanolamine solution was injected as a blocking agent. For pharmacokinetic measurements, diluted to 100, 50, 25, 12.5, 6.25, and 3.125 nM were used and injected sequentially from low concentration. Injected for 5 minutes at a flow rate of 10 μl/min to bind, and dissociated by injecting a running buffer for 30 minutes. The equilibrium dissociation constant, K _D value from the surface plasmon resonance measurement was calculated as _{k off} /k _on. Biacore data is summarized in Table 5.

Coupling constant
(k _on , 1/Ms) Dissociation constant
(k _off , 1/s) Affinity for antigen
(K _D , M) 4D5 2.09 x 10 ⁵ 8.82 x 10 ^-5 4.22 x 10 ^-10 D98W 3.97 x 10 ⁵ 8.89 x 10 ^-5 2.24 x 10 ^-10 AH06 8.80 x 10 ⁵ 4.42 x 10 ^-5 5.02 x 10 ^-11 AH16 4.04 x 10 ⁵ 1.20 x 10 ^-4 2.97 x 10 ^-10 A058 5.22 x 10 ⁵ 2.78 x 10 ^-4 5.33 x 10 ^-10 A091 6.44 x 10 ⁵ 6.88 x 10 ^-4 1.07 x 10 ^-9

As shown in Table 5, the literature (Resi et al (2002) 21 :. 851-862) affinity of D98W reported in variants, the K _D value of 422 pM K _D value of the parent antibody 4D5 with 224 pM Compared to this, the anti-ErbB2 antibody variant AH06 of the present invention exhibited an 8-fold improved affinity compared to the parent antibody 4D5 with a _{K D value of 50.2 pM.} In addition, AH16 of the present invention showed a K _D value of 297 pM, which was about 2 times improved compared to the parent antibody 4D5 (Table 5). These results indicate that the 4D5 variant of the present invention has significantly improved antigen affinity compared to the parent antibody 4D5.

Example 7. Anti- ErbB2 In vitro cell proliferation inhibitory activity test of antibody

In order to confirm the ErbB2-mediated disease inhibitory effect of each anti-ErbB2 antibody variant, a gastric cancer cell line NCI-N87 cells (ATCC Accession No. CRL-5822) were used to test the ability of the antibody to inhibit cell proliferation. For the evaluation of cell proliferation inhibitory activity, NCI-N87 cells were detached using 0.05% trypsin-EDTA ^{, suspended in a medium to a concentration of 0.75×10 4} cells/ml, and inoculated into 96-well cell culture plates by 0.1 ml each. . After inoculation, the cells were cultured to adhere to the cells at 37° C. and 5% CO _{2, and the cells were treated with humanized antibodies.} Antibodies were treated at concentrations of 10, 1, 0.1, 0.01 and 0.001 µg/ml, and RPMI1640/2% FBS medium was used for antibody treatment. After reacting for about 6 days, WST-8 (Dojindo) was treated to perform a color reaction for 3-4 hours, and a wavelength of 450 nm was measured.

The anti-ErbB2 antibody variant and the 4D5 antibody inhibiting cell proliferation activity against NCI-N87 cells are shown in Figs. 6-8. As shown in Figs. 7 and 8, the anti-ErbB2 antibody variant of the present invention increased cell proliferation inhibitory activity by about 3.5 times compared to the parent antibody 4D5. On the other hand, in Example 6, the cell proliferation inhibitory activity of D98W, which was measured to have an affinity improvement of about 2 times compared to the parent antibody 4D5, only showed the same level of activity as that of 4D5 (FIG. 6). _{Specifically, the IC 50} of A091 of the present invention is 10.3 nM (the IC ₅₀ of 4D5 is 25.4 nM), which is about twice as effective as 4D5, and the IC ₅₀ for A058 is 4.3 nM (the IC ₅₀ for 4D5 is 10.4 nM). ), the efficacy was improved by about 2.5 times compared to 4D5 (FIG. 7). In addition, the IC ₅₀ for AH06 was 3.4 nM (IC ₅₀ for 4D5 was 11.6 nM), which was about 3.5 times more effective than 4D5 (FIG. 8).

These results indicate that the 4D5 variants of the present invention have significantly improved not only affinity, but also cell proliferation inhibitory activity, compared to the parent antibody 4D5 and D98W disclosed in the literature.

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

<110> Chongkundang <120> anti-ErbB2 antibody variants <130> PN120220D <160> 20 <170> KopatentIn 2.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH amino acid sequence of 4D5 <400> 1 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 Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL amino acid sequence of 4D5 <400> 2 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 Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH amino acid sequence of 4D5 variant <400> 3 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 Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Trp Gly Phe Tyr Ala Phe Ala Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH amino acid sequence of 4D5 variant <400> 4 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 Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Asn Ala Lys Gly Phe Tyr Ser Phe Val His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL amino acid sequence of 4D5 variant <400> 5 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 Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Gln Thr Pro Ala 85 90 95 Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 6 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH amino acid sequence of 4D5 variant <400> 6 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 Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Arg Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Trp Gly Phe Tyr Ala Phe Ala Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL amino acid sequence of 4D5 variant <400> 7 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 Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Thr Thr Thr Trp Pro Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Thr Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> linker sequence <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ser 1 5 10 <210> 9 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> LN01_R primer <400> 9 actagtgcta ctcacggtca ccagagttcc ctgtccccag taatccatgg cgtamnngcc 60 mnnmnnmnnm nntctagagc agtagtac 88 <210> 10 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> LN02_R primer <400> 10 actagtgcta ctcacggtca ccagagttcc ctgtccccam nnmnnmnnmn ngtagaagcc 60 atcacc 66 <210> 11 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> LN03_F primer <400> 11 gacttcgcta cgtactactg cnnknnknnk nnkaccactc ctccgac 47 <210> 12 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> LN04_F primer <400> 12 gacttcgcta cgtactactg ccaacagcac tacnnkactn nknnknnktt cggacaaggc 60 ac 62 <210> 13 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> LN05_R primer <400> 13 tgaatctaga tggcacaccm nnmnnmnnga amnnmnnmnn gtagatcagc agcttc 56 <210> 14 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> LN06_R primer <400> 14 tgaatctaga tggcacaccm nnmnnmnncc amnnmnnmnn gtagatcagc agcttc 56 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LN0506_F primer <400> 15 ggtgtgccat ctagattcag tg 22 <210> 16 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> LN07_R primer <400> 16 actagtgcta ctcacggtca ccagagttcc ctgtccccag taatccatmn ngtamnngcc 60 mnnmnnmnnm nntctagagc agtagtac 88 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> LR primer <400> 17 gcgcgctact cacggtc 17 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> LF primer <400> 18 ggcccaggcg gccgatatcc agatgac 27 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HF primer <400> 19 gagctcatgg atatccagat gacccagag 29 <210> 20 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> HR primer <400> 20 gcgcgctact cacggtc 17

Claims (4)

(a) a light chain variable domain of SEQ ID NO: 7; And (b) an anti-ErbB2 antibody variant comprising the heavy chain variable domain of SEQ ID NO: 6, or an antigen-binding fragment thereof.
A nucleic acid molecule encoding a heavy chain variable domain of an anti-ErbB2 antibody variant of claim 1 or an antigen-binding fragment thereof.
A nucleic acid molecule encoding a light chain variable domain of the anti-ErbB2 antibody variant of claim 1 or an antigen-binding fragment thereof.
(a) a pharmaceutically effective amount of the anti-ErbB2 antibody variant of claim 1 or an antigen-binding fragment thereof; And (b) a pharmaceutically acceptable carrier.

Images