KR20200098831A - Novel site-specific antibody fragment platform - Google Patents

Abstract

The present invention relates to a Fab fragment specifically binding to human epidermal growth factor receptor 2 (HER2), which comprises: (a) a heavy chain variable region (V_H) including an amino acid sequence of SEQ ID NO: 5; (b) a heavy chain constant region 1 (C_H1) including an amino acid sequence of SEQ ID NO: 6; (c) a light chain variable region (V_L) including an amino acid sequence of SEQ ID NO: 11; and (d) a site-specific light chain constant region (sC_L) including an amino acid sequence of SEQ ID NO: 15. A novel site-specific antibody fragment platform of the present invention enables mass production and homogenization of site-specific mono-valent or di-valent Fab fragments which can replace an existing antibody drug through E. coli, and provides binding affinity for HER2 similar or superior to Herceptin (trastuzumab), and thus can be useful as a technology for producing antibody-drug conjugates in the fields of medicine and pharmaceuticals.

Description

Novel site-specific antibody fragment platform {Novel site-specific antibody fragment platform}

The present invention relates to a novel site-specific antibody fragmentation platform, and more particularly, various molecules in the light chain constant region (C _L ) of a fragment antigen-binding (Fab), which is an antigen-binding determining site that specifically binds to HER2. It relates to a platform for the production of mono-valent antibody fragments or di-valent antibody fragments in which cysteine residues useful for conjugation are site-specifically substituted.

Antibody therapy has been established for targeted treatment of patients with cancer, immune diseases and angiogenic disorders [Carter, P., (2006) Nature Reviews Immunology 6:343-357]. Antibody-drug conjugates (ADCs) for local delivery to cytotoxic agents or cell proliferation inhibitors are immune conjugates that are used in the treatment of cancer by the use of drugs that kill or inhibit tumor cells. ) And to accumulate it in tumor cells [Junutula, et al., 2008b Nature Biotech. 26(8):925-932; Dornan, et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO2009/052249; McDonagh, (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina, et al., (2006) Bioconj. Chem. 17:114-124; Erickson, et al., (2006) Cancer Res. 66(8):1-8; Sanderson, et al., (2005) Clin. Cancer Res. 11:843-852; Jeffrey, et al., (2005) J. Med. Chem. 48:1344-1358; Hamblett, et al., (2004) Clin. Cancer Res. 10:7063-7070].

Cytotoxic drugs in antibody-drug conjugates are typically in a non-site-specific manner through the lysine side chain to provide an activated natural cysteine sulfhydryl group, or in an interchain disulfide present in the antibody. bond, -SS-) is conjugated to the antibody by reduction. With regard to the drug antibody ratio (DAR) and site of attachment, site-specific conjugation of drugs to antibodies is required to provide high homogeneity and batch-to-batch consistency to the antibody-drug conjugate population. Was considered. Site-specific attachment is typically achieved by substituting a natural amino acid in an antibody with an amino acid such as cysteine, to which a drug moiety can be conjugated [Stimmel, et al., (2000) JBC, 275(39):30445 -30450, conjugation of an IgG S442C variant with bromoacetyl-TMT; Junutula, et al., Nature Biotechnology 26(8):925-932]. In addition, in the prior literature [Jujuntula, et al.], a site-specific antibody-drug conjugate in which a drug moiety is attached to a specific cysteine residue engineered with an antibody sequence is non-specifically conjugated to have similar efficacy compared to the antibody-drug conjugate. And reported to reduce systemic toxicity.

The composition of the antibody is composed of an antigen binding site, an antigen binding fragment (Fab) and an effect site F _C (crystallizable fragment, effector function), so that one Fab is bound to the effector as a dimer, so that it is di-valent. It has an antigen-binding site of The advantage of the dimer is that it has stability, but it is produced and used in mammalian cells, not in E. coli, as the sugar chain structure of the effect part. Therefore, in order to increase the efficiency and stability of these Fabs, two Fabs are linked (Fab) when an antibody that does not require binding to a sugar chain structure and complement preferentially requires only the antigen-binding portion (Fab) from which the Fc region has been removed. _It is necessary to develop a technology that can produce ₂ .

Human epidermal growth factor receptor (HER/EGFR/ERBB) family that receives extracellular signals. Human EGFR2 (HER2) is known as HER2/neu or ErbB2. It is a site that transmits intracellular signals and is a tyrosine kinase receptor (tyrosine). kinase receptor). It shows a very high structural similarity rate with the other three EGFR families (HER1, HER3, HER4). Although there is no known ligand that transmits a signal that binds to HER2, it forms a heterodimer by partnering with other HER receptors that bind to the ligand, thereby increasing the cell cycle, regulating cell proliferation and differentiation through various signaling. It is known to be involved in survival.

The EGFR family HER2 (Human epidermal growth factor receptor-2) has a close relationship with breast cancer. About 25% of breast cancer patients have a phenomenon in which the HER2 gene is proliferated and overexpressed according to the proliferation of cancer cells, which is classified as HER2-positive cancer, and more than that of HER2-negative cancer. It has been reported that the risk of recurrence or death is remarkably high. In HER2-positive breast cancer, a cell signaling process that promotes cancer growth by forming a dimer by tyrosine kinase by HER2 overexpressed has been revealed.

HER2 dimerization inhibitor Perjeta [Perjeta (Pertuzumab, Pertuzumab)], HER2 target anticancer drug Herceptin [Herceptin (trastuzumab)], HER2-positive metastatic breast cancer secondary treatment Kesila [Kadcyla (trastuzumab)] Emtansine, Trastuzumab emtansine), etc., used in combination with capecitabine and Lapatinib, which works only in HER2-positive breast cancer, has appeared, and has been approved for the treatment of HER2-positive breast cancer. In addition, as well as breast cancer, colon cancer, gallbladder cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, uterine cancer, pancreatic cancer, prostate cancer, brain cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer , As overexpression of the HER2 gene has been observed in esophageal cancer and gastric cancer, recently, HER2-related anticancer drugs are used in combination in lung cancer and gastric cancer, or related treatment studies are underway.

Herceptin (trastuzumab) as a HER2-positive anticancer drug It is a humanized monoclonal antibody for recombinant humanized therapy targeting the extracellular domain of HER2 protein. Herceptin (trastuzumab) is effective only when the gene protein HER2 receptor is positive. It is reported that the recurrence rate decreases by 50% and the mortality rate by 30% when administered for 1 year in early breast cancer as well as relapsed/hypermetastatic breast cancer. It is evaluated as an important drug for the development of therapeutic agents.

There are two main mechanisms of action as anticancer agents of antibody therapeutics: ① Antigen neutralization by antigen binding ② Effector function (for example, antibody-dependent cytotoxicity and complement-dependent cytotoxicity) Function) through the activation of the immune system. However, in the case of Herceptin (trastuzumab), it exhibits anticancer activity through inhibition of human HER2 corresponding to its antigen, and the effect function is not important, so the same effect can be expected even by removing the Fc region that mediates the effect function of the antibody. Therefore, if the binding force to HER2 is maintained, a fragment of Herceptin (trastuzumab) can exhibit similar anticancer efficacy.

Therefore, Fab-type fragments are produced using E. coli and can be expected to have an economical effect that can replace Herceptin (trastuzumab), and they work with the same mechanism that binds to human HER2 and inhibits the dimer of HER2. Can have the same efficacy in In addition, since the antibody fragment improved by fragmenting the antibody is smaller in size compared to the intact antibody, the penetration rate into tissues or tumors is good, and low-cost production is possible using a bacterial system. However, since antibody fragments do not have Fc and have a smaller molecular weight compared to intact antibodies, there is a disadvantage that the half-life in the blood may be shortened due to inhibition of antibody recycling and increase in kidney excretion, but functional peptide fusion and PEGylation Various biopharmaceutical stabilization technologies are being developed, including such as the case of Cimzia approved by the FDA. In addition, currently marketed antibody-drug conjugate drugs include gemtuzumab ozogamicin, brentuximab vedotin, and trastuzumab emtansine, but all of the above drugs It is a drug developed based on the entire antibody region, not the antibody fragment (Fab). Accordingly, there is a need to develop a technology for producing antibody-drug conjugates that can develop antibody fragments (Fab)-based drugs.

Korean Patent Registration No. 10-1453462 (2014.10.15) Korean Patent Application Publication No. 10-2018-0039919 (2018.04.19) Korean Patent Application Publication No. 10-2016-0127317 (2016.11.03) Korean Patent Application Publication No. 10-2015-0112385 (2015.10.07)

The inventors of the present invention were studying for the production of antibody-drug conjugates based on antibody fragments that can enhance the safety and efficacy of antibody-drug conjugates, while a cysteine residue was introduced mono-valent- or divalent- It was found that by producing (di-valent) site-specific point mutants, a novel site-specific Fab platform can be mass-produced through E. coli and can be produced in a homogeneous form. In addition, it was confirmed that the novel site-specific monovalent- or divalent Fab fragment specifically binds to HER2 due to excellent binding affinity.

Accordingly, an object of the present invention is to provide a novel site-specific antibody fragmentation platform.

According to an aspect of the present invention, there is provided a fragment antigen-binding (Fab) fragment that specifically binds to human epidermal growth factor receptor 2 (HER2) and includes the following regions: (a) the amino acid sequence of SEQ ID NO: 5 A heavy chain variable region comprising a (V _H ); (b) heavy chain constant region 1 (C _H1 ) comprising the amino acid sequence of SEQ ID NO: 6; (c) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 (V _L ); And (d) a site-specific light chain constant region comprising the amino acid sequence of SEQ ID NO: 15 (sC _L ).

In one embodiment, the Fab fragment may be a mono-valent antibody fragment or a di-valent antibody fragment.

In one embodiment, the divalent (di-valent) antibody fragment comprises a linker comprising the amino acid sequence of SEQ ID NO: 17 at the C-terminus of the light chain constant region (C _L ) and a SARAH domain comprising the amino acid sequence of SEQ ID NO: 18 It may be additionally included. In addition, the di-valent antibody fragment may be recombined through hydrophobic bonds between SARAH domains.

According to another aspect of the present invention, there is provided an expression construct comprising the following for preparing a Fab fragment that specifically binds to human epidermal growth factor receptor 2 (HER2): (a) the nucleotide sequence of SEQ ID NO: 8 A heavy chain variable region comprising a (V _H )-encoding nucleic acid molecule and a heavy chain constant region 1 (C _H1 ) comprising a nucleotide sequence of SEQ ID NO: 9-encoding a heavy chain-expressing construct comprising a nucleic acid molecule; And (b) a light chain variable region (V _L )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 13 and a site-specific light chain constant region (sC _L )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 16 A light chain-expression construct comprising.

According to another aspect of the present invention, a recombinant vector comprising the expression construct is provided.

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

In one embodiment, the host cell may be E. coli.

According to another aspect of the present invention, (i) culturing the host cell; (Ii) expressing a Fab fragment comprising a site-specific light chain constant region (sC _L ) in the cultured host cell; And (iii) comprising the step of recombining the expressed Fab fragment, there is provided a method for producing a Fab fragment that specifically binds to HER2.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer comprising the Fab fragment.

In one embodiment, the cancer is breast cancer, colon cancer, gallbladder cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, uterine cancer, pancreatic cancer, prostate cancer, brain cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, liver cancer , Lung cancer, head and neck cancer, esophageal cancer, and may be one selected from the group consisting of gastric cancer.

According to the present invention, the site-specific Fab fragment specifically binds to HER2, and may be one Fab fragment (a monovalent antibody fragment) or two Fab fragments (divalent antibody fragment) bound through the SARAH domain. It turned out. In addition, it was found that site-specific Fab fragments can be prepared homogeneously through E. coli. The site-specific Fab fragment according to the present invention has similar or superior binding affinity for HER2 compared to Herceptin (trastuzumab) and is useful for diagnosis, prevention and treatment of breast cancer.

Therefore, the novel site-specific antibody fragmentation platform of the present invention can be usefully used as a technology for producing antibody-drug conjugates in the fields of medicine and pharmaceuticals.

1 is a schematic diagram of fragments of the site-specific (a) and non-site-specific (b) antibodies of the present invention.
Figure 2 is a construct for each domain-specific antibody fragment domain of the present invention.
3 is a schematic diagram of a re-constitutional site-specific antibody fragment construct of the present invention.
4 is a result of the expression pattern of the site-specific antibody fragment in E. coli.
5 is a result showing the denaturation (denaturation) of the inclusion body of the antibody fragment produced in E. coli.
6 is a result of purification through recombination of site-specific antibody fragments.
7 is a result of confirming the homogeneity of the site-specific antibody fragment [(a) APm914, (b) APd924, (c) APm208 and (d) APd208].
8 is a result of binding of the site-specific antibody fragment to the actual DBCO-maleimide group.
9 is a result of comparing the binding affinity of the control drug Herceptin (trastuzumab) and the site-specific antibody fragment to the antigen (HER2).

The present invention provides a fragment antigen-binding (Fab) fragment that specifically binds to human epidermal growth factor receptor 2 (HER2) and includes the following regions: (a) a heavy chain variable comprising the amino acid sequence of SEQ ID NO: 5 Region (V _H ); (b) heavy chain constant region 1 (C _H1 ) comprising the amino acid sequence of SEQ ID NO: 6; (c) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 (V _L ); And (d) a site-specific light chain constant region comprising the amino acid sequence of SEQ ID NO: 15 (sC _L ).

"Fab fragment" refers to a fragment having an antigen-binding function, and includes a heavy chain variable region (V _H ), a heavy chain constant region 1 (C _H1 ), a light chain variable region (V _L ), and a light chain constant region (C _L ). Branches are structured and have one antigen binding site. Such Fab fragments can be obtained using proteolytic enzymes [for example, restriction cleavage of the whole antibody with papain yields Fab, and cleavage with pepsin yields F(ab') ₂ fragments], preferably It can be produced through genetic recombination technology.

“Heavy chain” refers to a full-length heavy chain comprising a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 and fragments thereof comprising an amino acid sequence with sufficient variable region sequence to confer specificity to the antigen. Means all. The Fab fragment in the present invention is a Fab fragment comprising a heavy chain composed of V _H and C _H1 .

The "light chains" means both a full-length light chain and fragments thereof containing the variable region domains V _L and C _L constant region domain comprises an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen. The Fab fragment in the present invention is a site-specific Fab fragment in which the 20th amino acid serine (Ser), which is a site necessary for site-specific conjugation, is substituted with cysteine (Cys) in the light chain consisting of the V _L and C _L.

The site-specific Fab fragment of the present invention may contain a variant of the amino acid sequence described in the appended sequence listing within the range capable of specifically binding to HER2. For example, a change can be made to the amino acid sequence of the Fab fragment to improve the binding affinity and/or other biological properties of the Fab fragment. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the Fab segment. 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 a mutation, the hydropathy index of the amino acid 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 similar biological activities can be retained only by substitution with amino acids having a similar hydrophobicity index. When introducing a mutation with reference to the hydrophobicity index, substitutions are made between amino acids having a hydrophobicity index difference of 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 showing substantial identity to the sequence listed in the sequence listing. The above substantial identity is at least 61% when the sequence of the present invention and any other sequence described above 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 showing 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 prior literature [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., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. 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.

The site-specific Fab fragment that specifically binds to HER2 of the present invention is a heavy chain and a light chain monomer or heterodimer.

In addition, the present invention includes one or two Fab fragments, wherein the site-specific Fab fragment is characterized in that a dimer is formed through hydrophobic binding between a monomeric monovalent antibody fragment and a SARAH domain. This is for the di-valent antibody fragment. By the SARAH domain, the two Fab fragments form a polymer in the opposite direction to each other, do not insert the amino acid'EC' sequence of the light chain constant region (C _L ) domain, and a linker (GGGS, SEQ ID NO: 17) and SARAH at the ends thereof. By inserting a domain (SEQ ID NO: 18), the effect of antigen binding on folding can be minimized.

The SARAH domain may form dimerization by two monomers. Each SARAH domain contains two helixes, forming an antiparallel helix dimer, and undergoing a head-to-tail interaction to form a dimer. The dimer interface is stabilized through hydrophobic interactions.

In addition, the present invention provides an expression construct comprising the following for preparing a Fab fragment that specifically binds to HER2: (a) a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO: 8 (V _H )- A heavy chain-expression construct comprising an encoding nucleic acid molecule and a heavy chain constant region 1 (C _H1 )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 9; And (b) a light chain variable region (V _L )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 13 and a site-specific light chain constant region (sC _L )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 16 A light chain-expression construct comprising.

In order to prepare a di-valent site-specific antibody fragment, the linker-encoding nucleic acid molecule may include a nucleotide sequence represented by SEQ ID NO: 19, and the SARAH domain-encoding nucleic acid molecule is represented by SEQ ID NO: 20. It may contain a nucleotide sequence.

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 sites are modified. Also included are known analogs [Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)].

Nucleic acid molecule sequences encoding heavy chain variable regions and light chain variable regions of the Fab fragment of the present invention can be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.

It is interpreted that the nucleic acid molecule encoding the antibody of the present invention also includes a nucleotide sequence exhibiting substantial identity only 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. Of homology, more preferably at least 90% of homology, and most preferably at least 95% of homology.

The expression construct produced in the present invention is constructed so as to express a desired gene in a host cell. In general, promoters and terminators operably linked to upstream and downstream of the expression construct are located, respectively. “Promoter” means a DNA sequence that regulates the expression of a coding sequence or functional RNA. In the recombinant vector of the present invention, the nucleotide sequence of interest is operably linked to the promoter. “Operatively linked” means a functional linkage between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcriptional control 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.

In addition, the present invention provides a recombinant vector comprising the expression construct.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are in the prior literature [Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001)]. It is disclosed. 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 prokaryotic cells as a host. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression.

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 proceeding transcription (eg, T7 promoter, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL λ Promoter, pR λ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter and trp promoter, etc.), ribosome binding site for initiation of translation and transcription/translation termination sequence (terminator such as T7 terminator, ADH1 terminator, T3 Terminators and TonB terminators, etc.). When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway [Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)] and the left-handed promoter of phage λ (pL λ promoter, Herskowitz) , I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as regulatory sites.

Vectors that can be used in the present invention include plasmids often used in the art (e.g., pACYCDuet-1, pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phage (e.g., λλλλΔ and M13, etc.) Or it can be produced by manipulating a virus (eg, SV40, etc.).

In addition, the present invention provides a host cell transformed with the recombinant vector.

In addition, the present invention comprises the steps of (i) culturing the host cell; (Ii) expressing a Fab fragment comprising a site-specific light chain constant region (sC _L ) in the cultured host cell; And (iii) it provides a method for producing a Fab fragment that specifically binds to HER2, comprising the step of recombining the expressed Fab fragment.

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, E. coli C43 (DE3), E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis strains of the genus Bacillus, and Salmonella typhimurium , Serratia Marsesons and various Pseudomonas species such as enterobacteriaceae and strains.

The method of transforming the vector of the present invention into a host cell is a heat shock method, a CaCl ₂ method [Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)], one method [Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); And Hanahan, D., J. Mol. Biol., 166:557-580 (1983)] and electroporation methods [Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)] and the like.

In addition, the present invention provides a pharmaceutical composition for cancer treatment comprising the Fab antibody fragment.

In one embodiment, the cancer is breast cancer, colon cancer, gallbladder cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, uterine cancer, pancreatic cancer, prostate cancer, brain cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, liver cancer , Lung cancer, head and neck cancer, esophageal cancer, and may be one selected from the group consisting of gastric cancer.

When the site-specific Fab fragment of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, silicic acid. Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It is not. 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 is preferably applied by parenteral administration.

A suitable dosage of the pharmaceutical composition of the present invention can be variously prescribed depending on factors such as the method of formulation, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient. I can. A typical dosage of the pharmaceutical composition of the present invention is in the range of 0.001 μg/kg-1000 mg/kg on an adult basis.

The pharmaceutical composition of the present invention is prepared in a 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 Alternatively, it may be manufactured by placing it in a multi-volume container. At this time, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. HER2 target antibody fragment construct construction

In order to develop a therapeutic agent and a diagnostic agent for HER2-positive breast cancer, a construct of an antibody fragment, Fab, was prepared. The amino acid sequence was determined so that a total of 4 kinds of candidate material constructs could be expressed in E. coli, and based on this, the DNA sequence was requested to Cosmo Genetech for synthesis. A candidate material construct was produced by cloning using the synthesized DNA sequence.

1) APm201 and APm202 constructs (APm201: V _H , C _H1 +6His, APm202: V _L , C _L )

(1) APm201 is a domain including V _H (heavy chain variable region: heavy chain variable region) and C _H1 (heavy chain constant region 1: heavy chain constant region 1) among domains constituting an antibody, and a disulfide bond present in the domain (intra-chain disulfide bond) stabilizes the structure of each domain. Two cysteines present in the hinge domain form a disulfide bond of each domain and play an important role in forming a dimer of each domain. However, when the hinge domain is deleted, a Fab fragment is formed. And can produce mono-valent Fab. In addition, cysteine may be linked in the amino acid'EC' sequence for the C _L domain of the Fab fragment and the amino acid'CDK' sequence for the C _H1 domain to play an important role in forming a Fab. However, cysteine may interfere with the specific-site binding of the drug and diagnostic agent in the binding between the maleimide group and cysteine in order to produce a therapeutic agent and a diagnostic agent combined with a drug and a diagnostic agent. Therefore, it was removed to produce a construct (see Fig. 1 and Tables 2 to 6). These cysteine-removed V _H +C _H1 and V _L +C _L are produced as inclusion bodies in the cytoplasm when produced in E. coli, respectively, and each domain is injected into one cell to express a large amount of protein. Made it possible.

(2) Based on the amino acid sequence of Trastuzumab (see Table 1) to name the antibody fragmentation platform, the unit-cost Fab, as APm and clone the V _H +C _H1 and V _L +C _L domains of the Fab, respectively. Thus, the DNA nucleotide sequence for the amino acid sequence of V _H +C _H1 and V _L +C _L (see Table 2 and Table 3) was requested from Cosmo Genetech to proceed with the synthesis. The underlined portions of the sequences of Tables 1 to 6 indicate variable regions. Using this, PCR primers (see Table 7) were prepared at the sites corresponding to the V _H +C _H1 and V _L +C _L domains, and the genes were cloned to express V _H +C _H1 and V _L +C _L , respectively. , This was cloned into the pET21-a vector (Novagen), which is an E. coli expression vector by treatment with a restriction enzyme.

<Amino acid sequence and base sequence of a control treaty (trastuzumab, Trastuzumab)>

<APm201 amino acid sequence and base sequence>

<APm202 amino acid sequence and base sequence>

<APm204 amino acid sequence and base sequence>

<APm904 amino acid sequence and base sequence>

<APm909 amino acid sequence and base sequence>

2) APm204 construct (APm204: V _L , C _L +GGGS+SARAH)

(1) APm204 removes variable dimerization by disulfide bonds by inserting the SARAH domain, a dimerization domain, into the V _L -C _L (light chain constant region) domain of the antibody constituent domain at the C-terminus It is one stable homodimer. In addition, the construct is a construct for a di-valent Fab that does not inhibit the binding ability to antigens with each other by duplexing in the reverse direction as well as stability. In order to form a reverse polymer by the SARAH domain, the amino acid'EC' sequence of the C _L domain was not inserted, but the'GGGS (4 amino acids)' sequence was inserted at the end, followed by inserting the SARAH domain, folding To minimize the effect of antigen binding to (see Table 4).

Thereby, in order to produce a therapeutic or diagnostic agent by conjugating a drug or a diagnostic agent, a construct by removing a residue capable of forming a non-specific site binding in a maleimide group and cysteine binding reaction Was produced. In order to clone the construct, it was converted into a codon capable of expressing E. coli, and the DNA sequence (see Table 4) was requested to Cosmo Genetech to proceed with synthesis. As a template, PCR primers for the corresponding site (see Table 7) were prepared, and each of the V _L +C _L -SARAH into which the SARAH domain was inserted was cloned to be expressed as an inclusion body in the cytoplasm when produced in E. coli. Each construct was injected into one cell and expressed so that a large amount of protein could be obtained.

3) Preparation of APm904 and APm909 constructs (APm904: APm202-S127C, APm909: APm204-S127C)

(1) APm904 (monomer, mono-valent, see Table 5) and APm909 (dimer, di-valent, see Table 6) were previously known Herceptin (Tra) for constructing constructs for site-specific conjugation. Stuzumab) is a point mutant of the V _L -C _L domain in which the 127th serine residue in the light chain is replaced with a cysteine residue required for disulfide bonds based on the molecular structure of the antibody fragment. In order to produce this, APm202 and APm204 were produced using primers for making point mutants (see Table 7) as templates.

Construct Primer Sequence (5' → 3') APm201_F(Nde1): ggaattcCATATGGAAGTTCAGCTGGTTGAATC APm201_R(Xho1): ccgCTCGAGGCTTTTCGGCG APm202_F(Nde1) ggaattcCATATGGATATTCAGATGACCCAGAGCC APm202_R(stop-Xho1) ccgCTCGAGCTAGCCGCGGTTAAAGC APm204_Lc_F(Nde1) ggaattcCATATGGATATTCAGATGACCCAGAGCC APm204_Sarah_R(stop-Xho1) ccgCTCGAGCTATTTAGCGTCCATCG APm904_F (Mutant) GAGCGATGAACAGCTGAAATGCGGCACCGCGAGCGTGGTGTGC APm904_R (Mutant) GCACACCACGCTCGCGGTGCCGCATTTCAGCTGTTCATCGCTC

2. Confirmation of E. coli expression of each construct (APm201, APm202, APm204, APm904 and APm909)

In order to check the protein expression of each construct gene, BL21 (DE3, pLysS) cells, which are E. coli-expressing cell lines, were transformed through heat shock at 42°C. One colony was inoculated into LB medium, cultured at 37° C. for 7 hours, treated with 0.5 mM IPTG, and cultured at 37° C. for 4 hours, and the expression was confirmed through 12% SDS-PAGE.

As a result of expression, it was confirmed that all constructs were each overexpressed (see FIG. 4).

3. Culture and purification of antibody fragments

1) Culture and purification of antibody fragments APm201, APm202, APm204, APm904 and APm909

The host cell for purification of the antibody fragment was BL21 (DE3, pLysS), an E. coli for T7 polymerase overexpressing recombinant protein, and the plasmid was used to express each domain using a pET21 vector, and each inclusion body in the cytoplasm. body) to generate antibody fragments.

The medium used for seed culture to produce each protein is Lysogeny Broth (LB), and one colony is inoculated into LB medium containing ampicillin, and 150 rpm using a shaking incubator at 37°C. Incubated overnight. Then, inoculate approximately 0.5 to 2% of cell culture medium in 1L TB medium (Gellix) containing new ampicillin, and incubate with shaking at 150 rpm for 5 hours, and then treatment with 0.2 mM IPTG when the OD ₆₀₀ value reaches between 1 and 2 Then, it was further incubated for 5 hours at 37°C and 150 rpm. After the culture was completed, the culture solution was centrifuged at 4° C. and 6000 rpm to obtain a pellet. The cells thus recovered were stored and used at -80°C. About 80 ml of cell lysis buffer [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, pH 7.4] per 1 L of the culture medium was added to the cells expressing the antibody fragment, followed by mixing with the cells. Thereafter, the cells were lysed for 10 minutes (pulse on 1 second, pulse off 1 second) using an ultrasonic disruptor. In order to separate the antibody fragments, proteins and cells on the inclusion body, centrifugation was performed for 40 minutes at a speed of 4° C. and 10,000 rpm using a centrifuge.

To wash the inclusion body separated by centrifugation, suspend once in 150 ml of buffer [50 mM Tris pH 7.5, 500 mM NaCl, 0.05% Triton X-100] per 1 L based on the culture medium, and centrifuge to obtain a precipitate again. Centrifugation was performed for 40 minutes at a speed of 4° C. and 10,000 rpm. In order to melt the inclusion body containing the antibody fragment thus obtained, it was melted using a melting buffer [8 M urea pH 8.0] per 1 liter of cells, and then stabilized by incubation at 55° C. for 40 minutes and then not melted again. In order to remove the material, additional centrifugation was performed for 40 minutes at 4° C. and 10,000 rpm. 8 M from antibody fragments resulting in a molten form from the urea APm202, APm904 APm909 and was used for recombination with the state to the V _H -C _H1 domain fused in 8 M urea, APm201 is then subjected to purification V _L -C _It was used for recombination with _L.

Affinity chromatography was performed to purify only antibody fragments in an aqueous APm201 solution melted in 8 M urea. In an open column, an NI-NTA resin capable of binding to 6his at the C-terminus of APm201 was charged, and 5 CV (5) in a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol] Column volume) to equilibrate. After allowing the resin and antibody fragments to bind by flowing an aqueous solution of APm201, 5 CV of wash buffer (8 M urea pH 8.0) was poured, and then 5 CV of 4 M urea wash buffer [50 mM Tris pH 7.5, 500 mM NaCl , 4 M urea, 70 mM imidazole] to remove non-specifically bound impurities and lower the urea concentration. Elution buffer to separate the APm201 antibody fragment from the Ni-NTA resin [elution buffer; 50 mM Tris pH 7.5, 500 mM NaCl, 4 M urea, 500 mM imidazole] was flowed. As in the experimental results, as a result of expressing the APm201, APm904 and APm909 constructs in the pET21 vector, it was confirmed that the antibody fragment was expressed in the form of an inclusion body (see FIG. 5).

2) Production of APm208 through recombination of APm201 and APm202

For recombination of antibody fragments melted in urea, APm201 (V _H +C _H1 ) and APm202 (V _L +C _L ) are mixed in a ratio of 3: 1, and then diluted buffer solution so that the final urea concentration is 0.5 M or less. After suspending in [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol], at 4° C. for 3 days to induce hydrophobic binding between APm201 (V _H +C _H1 ) and APm202 (V _L +C _L ). It was reacted at 100 rpm.

In order to remove the precipitate of the solution reacted with APm201 (V _H +C _H1 ) and APm202 (V _L +C _L ), the precipitate was removed by centrifugation at 4° C. and 10,000 rpm for 20 minutes. In addition, in order to remove the unrecombined part to produce the aqueous APm208, NI-NTA resin that can bind to 6his at the C-terminus of APm201 is filled and a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, After equilibration with 5 CV of [10% glycerol], pour a solution of APm201 (V _H +C _H1 ) and APm202 (V _L +C _L ) to allow resin and APm201 to bind, and then 5 CV of washing buffer [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 70 mM imidazole] was poured to remove APm202, which did not bind to APm201. In order to separate the antibody fragments of APm201 and APm208 from the Ni-NTA resin, an elution buffer [elution buffer; 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 500 mM imidazole] was flowed. As can be seen from the experimental results, it was confirmed that APm208 was recombined through hydrophobic bonding in the solutions of APm201 and APm202 (see FIG. 6).

3) Production of APd212 through recombination of APm201 and APm204

For recombination of antibody fragments melted in urea, APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAH) were mixed in a ratio of 1: 3, and then diluted so that the concentration of the final urea was 0.5 M or less. After suspending in a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol], 3 days to induce hydrophobic binding between APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAH) During the reaction at 4° C. at 100 rpm.

In order to remove the precipitate of the solution obtained by reacting APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAH), the precipitate was removed by centrifugation at 4° C. and 10,000 rpm for 20 minutes. In addition, in order to remove the unrecombined part to produce the water-soluble APd212, NI-NTA resin that can bind to 6his at the C-terminus of APm201 (V _H +C _H1 ) was filled, and a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol] after equilibration with 5 CV and flowing a solution of APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAH), the resin and APm204 After allowing to bind, 5 CV of a washing buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 70 mM imidazole] was poured to remove APm204 not bound to APm201. In order to separate the antibody fragments of APm201 and APd212 from the Ni-NTA resin, an elution buffer [elution buffer; 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 500 mM imidazole) was flowed. As can be seen from the experimental results, it was confirmed that APd212 was recombined through hydrophobic bonding in the solutions of APm201 and APm204 (see FIG. 6).

4) Production of site-specific monovalent antibody fragment APm914 through recombination of APm201 and APm904

For recombination of antibody fragments melted in urea, APm201 (V _H +C _H1 ) and APm904 (V _L +sC _L ) are mixed in a ratio of 1: 3, and then diluted buffer solution so that the final urea concentration is 0.5 M or less. After suspending in [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol], at 4° C. for 3 days to induce hydrophobic bonding between APm201 (V _H +C _H1 ) and APm904 (V _L +sC _L ). It was reacted at 100 rpm.

In order to remove the precipitate of the solution reacted with APm201 (V _H +C _H1 ) and APm904 (V _L +sC _L ), the precipitate was removed by centrifugation at 4° C. and 10,000 rpm for 20 minutes. In addition, in order to remove the unrecombined part in order to produce the water-soluble APm914, NI-NTA resin that can bind to 6his at the C-terminus of APm201 is filled, and a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, After equilibration with 5 CV of [10% glycerol], flow a solution of APm201 (V _H +C _H1 ) and APm904 (V _L +sC _L ) to allow resin and APm201 to bind, and then 5 CV of washing buffer A solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 70 mM imidazole] was poured. In order to separate the antibody fragments of APm201 (V _H +C _H1 ) and APm914 from Ni-NTA resin, an elution buffer (elution buffer; 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 500 mM imidazole] was flowed. As can be seen from the experimental results, it was confirmed that the site-specific monovalent antibody fragment APm914 was recombined through hydrophobic binding in the solutions of APm201 (V _H +C _H1 ) and APm904 (V _L +sC _L ) (see FIG. 6). .

5) Production of site-specific bivalent antibody fragment APd924 through recombination of APm201 and APm909

For recombination of the antibody fragments melted in urea, APm201 (V _H +C _H1 ) and APm909 (V _L +sC _L +SARAH) were mixed in a ratio of 1: 3 and diluted so that the final urea concentration was 0.5 M or less. 3 days to induce hydrophobic binding between APm201 (V _H +C _H1 ) and APm909 (V _L +sC _L +SARAH) after suspending in a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol] During the reaction at 4° C. at 100 rpm.

In order to remove the precipitate of the solution obtained by reacting APm201 (V _H +C _H1 ) and APm909 (V _L +sC _L +SARAH), the precipitate was removed by centrifugation at 4° C. and 10,000 rpm for 20 minutes. In addition, in order to remove the unrecombined portion to produce APd924 in the aqueous phase, NI-NTA resin that can bind to 6his at the C-terminus of APm201 is filled, and a buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol] after equilibration with 5 CV and flowing a solution of APm201 (V _H +C _H1 ) and APm909 (V _L +sC _L +SARAH) to allow resin and APm201 to bind, then 5 CV of Washing buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 70 mM imidazole] was flowed. In order to separate the antibody fragments of APm201 (V _H +C _H1 ) and APd924 from the Ni-NTA resin, an elution buffer (elution buffer; 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 500 mM imidazole) was flowed. As can be seen from the experimental results, it was confirmed that the site-specific bivalent antibody fragment APd924 was recombined through hydrophobic binding in the solutions of APm201 (V _H +C _H1 ) and APm909 (V _L +sC _L +SARAH) (Fig. 6 Reference).

4. Confirmation of homogeneity of APm208, APd212 APm914 and APd924

Size exclusion chromatography was performed to confirm the homogeneity of the refolded and recombinant antibody fragments APm208, APd212 APm914 and APd924. A HiLoad 16/600 Superdex 200 pg column (GE Healthcare) was connected to the AKTA Prime FPLC system, and an equilibration buffer (PBS pH 7.2) was flowed to bring it to equilibrium. An aqueous solution of antibody fragments eluted during the recombination process was flowed through the column, and retention volumes were measured to be about 85 ml for monovalent fragments APm208 and APm914, and about 68 ml for bivalent fragments APd212 and APd924. From the 280 nm absorbance indicating the antibody fragment in the chromatogram and the results of SDS-PAGE experiments, it was confirmed that the homogeneity of the antibody fragment was very high (see FIG. 7).

5. Confirmation of DBCO-maleimide binding to site-specific antibody fragments

The site-specific antibody fragments APm914 (mono-valent) and APd924 (di-valent) dissolved in PBS, and the control antibody fragments APm208 (mono-valent) and APd212 (di-valent) were prepared at 100 μM, respectively. A stock solution was prepared by dissolving DBCO-maleimide in a DBCO (dibenzocyclooctyne) solvent. After mixing the protein-to-DBCO-maleimide stock solution at a molar concentration of 1:4 ratio, it was reacted at room temperature for 1 hour. In order to confirm the binding of DBCO-maleimide to the site-specific antibody fragments APm914 and APd924, 5/6-carboxylrhodamine 110-PEG3-azide (5/6-Carboxylrhodamine) dissolved in DMSO in the above reaction product 110-PEG3-Azide) stock solution was mixed at a ratio of 1:4 relative to the protein molar concentration, and then reacted at room temperature for 30 minutes. The reaction was loaded on a 12% acrylamide gel and lowered by SDS-PAGE, and then the site-specific antibody fragment-DBCO_maleimide-5/6-carboxylodamine 110-PEG3-azide complex was confirmed with a fluorescent plate, Protein bands were further confirmed by Coomassie staining.

As a result, as it can be seen that fluorescence is displayed only in the bands of the light chain regions (V _L -sC _L ) of APm914 and PAd924 into which site-specific free-cysteine residues are inserted, DBCO-maleimide groups and monovalent and divalent sites- It was confirmed that conjugation of specific antibody fragments was performed (see FIG. 8).

6. sHER2-binding affinity measurement using enzyme-linked immunosorbent assay (ELISA)

100 ng/well of antigen sHER2 (exrecellular domain) was added to a 96-well ELISA plate and reacted overnight at 4° C. to immobilize the antigen on the plate surface. Then, the supernatant was removed and 200 µl of a blocking solution (Sigma, B6429-500ML) was added. It was dispensed into each well and blocked overnight at 4°C. Trastuzumab, a standard material for obtaining a calibration curve, and purified antibody fragments were diluted with PBS to a concentration of 0-125 ng/ml. Each 100 µl of each was dispensed and reacted at room temperature for 1 hour to bind to the antigen, and after the reaction was completed, washed three times with PBST (PBS, 0.05% tween 20, pH 7.4). Anti-human IgG (Sigma, I5260) was diluted 1/1,000 and dispensed into each well by 100 µl and reacted at room temperature for 1 hour, and then the supernatant was removed and washed 3 times with PBST. Secondary antibody anti-goat IgG-peroxidase (Sigma, A5420) was diluted 1/3,000, dispensed into each well by 100 μl, and reacted at room temperature for 1 hour. The supernatant was removed and washed 3 times with PBST, and then 100 μl of TMB color development reagent was dispensed. The reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ to the colored wells, and the results of measuring absorbance at 450 nm wavelength using a microplate reader are shown in FIG. 9.

As a result of converting the binding affinity to trastuzumab as a percentage, it was found that the site-specific monovalent APm914 was 104% and the divalent APd924 was 94.3%, which showed that the activity of the site-specific antibody fragment against HER2 was sufficient. It means to be maintained.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Novel site-specific antibody fragment platform <130> P190127 <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Anti-HER2 Light chain <400> 1 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 Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Anti-HER2 Heavy chain <400> 2 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 Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 3 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Anti-HER2 Light chain <400> 3 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgcc gcgcgagcca ggatgtgaac accgcggtgg cgtggtatca gcagaaaccg 120 ggcaaagcgc cgaaactgct gatttatagc gcgagctttc tgtatagcgg cgtgccgagc 180 cgctttagcg gcagccgcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240 gaagattttg cgacctatta ttgccagcag cattatacca ccccgccgac ccttggccag 300 ggcaccaaag tggaaattaa acgcaccgtg gcggcgccga gcgtgtttat ttttccgccg 360 agcgatgaac agctgaaaag cggcaccgcg agcgtggtgt gcctgctgaa caacttttat 420 ccgcgcgaag cgaaagtgca gtggaaagtg gataacgcgc tgcagagcgg caacagccag 480 gaaagcgtga ccgaacagga tagcaaagat agcacctata gcctgagcag caccctgacc 540 ctgagcaaag cggattatga aaaacataaa gtgtatgcgt gcgaagtgac ccatcagggc 600 ctgagcagcc cggtgaccaa aagctttaac cgcggcgaat gc 642 <210> 4 <211> 1352 <212> DNA <213> Artificial Sequence <220> <223> Anti-HER2 Heavy chain <400> 4 gaagttcagc tggttgaatc tggcggcggt ctggttcaac cgggcggttc cctgcgcctg 60 tcttgcgcag cgtcaggctt taacatcaaa gatacctaca tccactgggt tcgccaggcg 120 ccgggtaaag gtctggaatg ggtggctcgc atctacccga ccaacggcta tacccgttat 180 gctgacagcg ttaaaggccg cttcaccatt agcgcggata ccagtaaaaa tacggcatac 240 ctgcaaatga actcgttacg cgcggaagac accgcggtgt actactgctc ccgctggggc 300 ggggatggct tctacgcaat ggattactgg ggtcagggca cgctggtcac agtctccagc 360 gcctcgacaa aaggcccgag cgtgttcccg ctggcgcctt ccagcaaaag cacctcaggg 420 ggcacagctg cgctgggttg cctggtgaaa gactacttcc cggaaccggt taccgtttcc 480 tggaacagcg gtgctctgac cagcggcgtt cacaccttcc cggctgttct gcaaagttcc 540 ggcctgtact ctctgagctc tgtggtgacc gtgccgtctt cttctctggg gacccagact 600 tacatttgca acgtgaacca caaaccgtca aacaccaaag ttgataaaaa agtggagccg 660 ccgaaaagct gcgataaaac ccatacctgc ccgccgtgcc cgggccggaa ctgctgggcg 720 gcccgagcgt gtttctgttt ccgccgaaac cgaaagatac cctgatgatt agccgcaccc 780 cggaagtgac ctgcgtggtg gtggatgtga gccatgaaga tccggaagtg aaatttaact 840 ggtatgtgga tggcgtggaa gtgcataacg cgaaaaccaa accgcgcgaa gaacagtata 900 acagcaccta tcgcgtggtg agcgtgctga ccgtgctgca tcaggattgg ctgaacggca 960 aagaatataa atgcaaagtg agcaacaaag cgctgccggc gccgattgaa aaaaccatta 1020 gcaaagcgaa aggccagccg cgcgaaccgc aggtgtatac cctgccgccg agccgcgatg 1080 aactgaccaa aaaccaggtg agcctgacct gcctggtgaa aggcttttat ccgagcgata 1140 ttgcggtgga atgggaaagc aacggccagc cggaaaacaa ctataaaacc accccgccgg 1200 tgctggatag cgatggcagc ttttttctgt atagcaaact gaccgtggat aaaagccgct 1260 ggcagcaggg caacgtgttt agctgcagcg tgatgcatga agcgctgcat aaccattata 1320 cccagaaaag cctgagcctg agcccgggca aa 1352 <210> 5 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 5 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> 6 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> CH1 <400> 6 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 Pro Lys Ser 100 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> C-terminal <400> 7 Leu Glu His His His His His His 1 5 <210> 8 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 8 gaagttcagc tggttgaatc tggcggcggt ctggttcaac cgggcggttc cctgcgcctg 60 tcttgcgcag cgtcaggctt taacatcaaa gatacctaca tccactgggt tcgccaggcg 120 ccgggtaaag gtctggaatg ggtggctcgc atctacccga ccaacggcta tacccgttat 180 gctgacagcg ttaaaggccg cttcaccatt agcgcggata ccagtaaaaa tacggcatac 240 ctgcaaatga actcgttacg cgcggaagac accgcggtgt actactgctc ccgctggggc 300 ggggatggct tctacgcaat ggattactgg ggtcagggca cgctggtcac agtctccagc 360 360 <210> 9 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> CH1 <400> 9 gcctcgacaa aaggcccgag cgtgttcccg ctggcgcctt ccagcaaaag cacctcaggg 60 ggcacagctg cgctgggttg cctggtgaaa gactacttcc cggaaccggt taccgtttcc 120 tggaacagcg gtgctctgac cagcggcgtt cacaccttcc cggctgttct gcaaagttcc 180 ggcctgtact ctctgagctc tgtggtgacc gtgccgtctt cttctctggg gacccagact 240 tacatttgca acgtgaacca caaaccgtca aacaccaaag ttgataaaaa agtggagccg 300 ccgaaaagc 309 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C-terminal <400> 10 ctcgagcacc accaccacca ccac 24 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 11 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 Leu Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> CL <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 100 105 <210> 13 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 13 gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60 attacctgcc gcgcgagcca ggatgtgaac accgcggtgg cgtggtatca gcagaaaccg 120 ggcaaagcgc cgaaactgct gatttatagc gcgagctttc tgtatagcgg cgtgccgagc 180 cgctttagcg gcagccgcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240 gaagattttg cgacctatta ttgccagcag cattatacca ccccgccgac ccttggccag 300 ggcaccaaag tggaaattaa a 321 <210> 14 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> CL <400> 14 cgcaccgtgg cggcgccgag cgtgtttatt tttccgccga gcgatgaaca gctgaaaagc 60 ggcaccgcga gcgtggtgtg cctgctgaac aacttttatc cgcgcgaagc gaaagtgcag 120 tggaaagtgg ataacgcgct gcagagcggc aacagccagg aaagcgtgac cgaacaggat 180 agcaaagata gcacctatag cctgagcagc accctgaccc tgagcaaagc ggattatgaa 240 aaacataaag tgtatgcgtg cgaagtgacc catcagggcc tgagcagccc ggtgaccaaa 300 agctttaacc gcggc 315 <210> 15 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> sCL <400> 15 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Cys 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 100 105 <210> 16 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> sCL <400> 16 cgcaccgtgg cggcgccgag cgtgtttatt tttccgccga gcgatgaaca gctgaaatgc 60 ggcaccgcga gcgtggtgtg cctgctgaac aacttttatc cgcgcgaagc gaaagtgcag 120 tggaaagtgg ataacgcgct gcagagcggc aacagccagg aaagcgtgac cgaacaggat 180 agcaaagata gcacctatag cctgagcagc accctgaccc tgagcaaagc ggattatgaa 240 aaacataaag tgtatgcgtg cgaagtgacc catcagggcc tgagcagccc ggtgaccaaa 300 agctttaacc gcggc 315 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 17 Gly Gly Gly Ser One <210> 18 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> SARAH <400> 18 Asp Phe Asp Phe Leu Lys Asn Leu Ser Leu Glu Glu Leu Gln Met Arg 1 5 10 15 Leu Lys Ala Leu Asp Pro Met Met Glu Arg Glu Ile Glu Glu Leu Arg 20 25 30 Gln Arg Tyr Thr Ala Lys Arg Gln Pro Ile Leu Asp Ala Met Asp Ala 35 40 45 Lys <210> 19 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Linker <400> 19 ggtggcggat cc 12 <210> 20 <211> 147 <212> DNA <213> Artificial Sequence <220> <223> SARAH <400> 20 gattttgact tcctgaaaaa cctgagttta gaagaactgc agatgcgtct gaaagcactg 60 gacccgatga tggaacgtga aatcgaagag ctgcgtcagc gttacaccgc caaacgtcag 120 ccgattctgg acgcgatgga cgctaaa 147

Claims (11)

Fragment antigen-binding (Fab) fragment that specifically binds to human epidermal growth factor receptor 2 (HER2) and contains the following regions:
(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 (V _H );
(b) heavy chain constant region 1 (C _H1 ) comprising the amino acid sequence of SEQ ID NO: 6;
(c) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 (V _L ); And
(d) a site-specific light chain constant region comprising the amino acid sequence of SEQ ID NO: 15 (sC _L ). The Fab fragment according to claim 1, wherein the Fab fragment is a mono-valent antibody fragment or a di-valent antibody fragment. According to claim 2, wherein the di-valent (di-valent) antibody fragment is a linker comprising the amino acid sequence of SEQ ID NO: 17 at the C-terminus of the light chain constant region (C _L ) and SARAH comprising the amino acid sequence of SEQ ID NO: 18 Fab fragment, characterized in that it further comprises a domain. The Fab fragment according to claim 2, wherein the di-valent antibody fragment is recombined through hydrophobic bonds between SARAH domains. An expression construct comprising the following to prepare a Fab fragment that specifically binds to human epidermal growth factor receptor 2 (HER2):
(a) a heavy chain variable region comprising the nucleotide sequence of SEQ ID NO: 8 (V _H )-encoding nucleic acid molecule and a heavy chain constant region 1 (C _H1 ) comprising the nucleotide sequence of SEQ ID NO: 9-heavy chain comprising the encoding nucleic acid molecule- Expression construct; And
(b) a light chain variable region (V _L )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 13 and a site-specific light chain constant region (sC _L )-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 16 Light chain-expression construct. A recombinant vector comprising the expression construct of claim 5. A host cell transformed with the recombinant vector of claim 6. The host cell of claim 7, wherein the host cell is E. coli. (I) culturing the host cell of claim 7;
(Ii) expressing a Fab fragment comprising a site-specific light chain constant region (sC _L ) in the cultured host cell; And
(Iii) recombining the expressed Fab fragment
A method for producing a Fab fragment that specifically binds to HER2 (human epidermal growth factor receptor 2) comprising a. A pharmaceutical composition for cancer treatment comprising the Fab fragment of any one of claims 1 to 4. The method of claim 10, wherein the cancer is breast cancer, colon cancer, gallbladder cancer, cholangiocarcinoma, bladder cancer, ovarian cancer, uterine cancer, pancreatic cancer, prostate cancer, brain cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, melanoma, endometrial cancer, cervical cancer, Anticancer pharmaceutical composition, characterized in that one selected from the group consisting of liver cancer, lung cancer, head and neck cancer, esophageal cancer and stomach cancer.

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