KR20200097158A - Novel di-valent antibody fragment platform - Google Patents

Abstract

The present invention is to prepare a Fab fragment capable of replacing an antibody and a novel di-valent Fab fragment. According to an embodiment of the present invention, the present invention relates to a Fab fragment which specifically binds to Her2. To this end, a SARAH domain is additionally included at a C-terminus of a heavy chain constant region (C_H1) two Fab fragments can be recombined in the reverse direction through the SARAH domain to form a dimer, thereby securing excellent antigen-binding abilities since binding to an antigen is not inhibited.

Description

Novel di-valent antibody fragment platform {Novel di-valent antibody fragment platform}

The present invention is a novel divalent antibody fragmentation platform to produce di-valent antibody fragments by introducing the amino acid sequence constituting the dimer into the Fab, which is a site that binds to the antigen, to induce dimer formation of the Fab fragment. For the platform. More specifically, a Fab fragment specifically binding to Her2 (Human epidermal growth factor receptor-2) and a di-valent antibody fragment binding the Fab fragment.

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

Human epidermal growth factor receptor (HER/EGFR/ERBB) family that receives extracellular signals. Human EGFR2 (HER2) is known as HER2/neu or ErbB2, and has a tyrosine kinase receptor as an intracellular signaling site. . It shows very high structural similarity with the other three EGFR families (HER1, HER3 and 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, and is involved in cell cycle increase, cell proliferation regulation, and differentiation and survival through various signaling. Is known.

Among the EGFR family, Her2 (Human epidermal growth factor receptor-2) has a close relationship with breast cancer. About 25% of breast cancer patients have HER2 gene proliferation and overexpression according to cancer cell proliferation, which is classified as HER2-positive cancer, and it is reported that the risk of recurrence or death is significantly higher than that of HER2-negative cancer. Her2 positive The cell signaling process that promotes cancer growth by forming a dimer by Her2 overexpressed in breast cancer has been revealed. HER2 dimerization inhibitor Perjeta [Perjeta (Pertuzumab, Pertuzumab)], HER2 target anticancer agent Herceptin [Herceptin (Trastuzumab)], HER2-positive metastatic breast cancer secondary treatment Kesila [Kadcyla (trastuzumab M Tanshin, Trastuzumab emtansine)] and capecitabine are used in combination with Lapatinib, which works only on HER2 positive breast cancer, and has been approved for the treatment of HER2-positive breast cancer. In addition to 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 for lung cancer and gastric cancer, or related therapeutic research is underway.

Herceptin (trastuzumab) as a Her2-positive anticancer agent 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 and mortality rate decrease by 50% and mortality rate by 30% when administered for 1 year in early breast cancer as well as recurrent hypermetastatic breast cancer. It is evaluated as an important drug for its development. The main mechanisms of action as anticancer agents of antibody therapeutics are largely two. ① Antigen neutralization by antigen binding ② Effector function (ADCC and CDC functions, etc.) are known to activate the immune system, but Herceptin (trastuzumab) ) Shows anticancer activity through inhibition of human Her2 corresponding to the antigen, and the effector function does not play an important role, so the same effect can be expected even by removing the Fc region that mediates the effector function of the antibody. If maintained, fragments of Herceptin (trastuzumab) can show similar anticancer efficacy.

Therefore, Fab-type fragments are produced using E. coli and can be expected to have an economic effect that can replace Herceptin (trastuzumab), and they work with the same mechanism that binds to human Her2 and inhibits Her2 dimer. Will 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 lower 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 various biopharmaceutical stabilization technologies including PEGylation, etc. Are being developed, and this is the case with the FDA-approved Cimzia.

The present inventors have tried to prepare a Fab fragment that can replace an antibody and a novel di-valent Fab fragment. As a result, a novel antibody fragment di-valent Fab platform that can be produced through E. coli and produced in a homogeneous form has been developed. As an example, a Fab fragment and a novel di-valent Fab fragment that specifically bind to Her2 having excellent binding affinity were developed.

In order to solve the above problems, the present invention is a di-valent antibody fragment having a dimer Fab fragment, wherein the Fab fragment is (a) a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 5 ( V _H ); (b) a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 6 (C _H1 ); (c) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 11 (V _L ); And (d) a light chain constant region (C _L ) comprising an amino acid sequence represented by SEQ ID NO: 12, and a linker and a SARAH domain are further included at the C-terminus of the heavy chain constant region (C _H1 ). Characterized di-valent antibody fragments are provided.

The linker may include an amino acid sequence represented by SEQ ID NO: 15.

The SARAH domain may include an amino acid sequence represented by SEQ ID NO: 16.

In addition, the present invention provides a di-valent antibody fragment comprising two or more of the Fab fragments, wherein the Fab fragment is recombined through hydrophobic bonds between SARAH domains.

In addition, the present invention is a Fab fragment expression cassette for a di-valent antibody fragment having a dimer Fab fragment, (a) a heavy chain variable region comprising the nucleotide sequence represented by SEQ ID NO: 8 (V _H ) -Encoding nucleic acid molecule; (b) a heavy chain constant region 1 (C _H1 )-encoding nucleic acid molecule comprising the nucleotide sequence represented by SEQ ID NO: 9; (c) a light chain variable region (V _L )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 13; And (d) a light chain constant region (C _L )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 14; Including, a linker to the 3'end of the heavy chain constant region 1 (C _H1 )-encoding nucleic acid molecule -Coding nucleic acid molecule and SARAH domain-encoding nucleic acid molecule provides a Fab fragment expression cassette, characterized in that it further comprises.

The linker-encoding nucleic acid molecule may include a nucleotide sequence represented by SEQ ID NO: 17.

The SARAH domain-encoding nucleic acid molecule may include a nucleotide sequence represented by SEQ ID NO: 18.

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

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

In addition, the present invention provides an anticancer pharmaceutical composition comprising the Fab fragment.

The cancer is breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer, small intestine cancer, endocrine cancer, It may be thyroid cancer, parathyroid cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer, or bone marrow cancer.

In addition, the present invention provides a method for producing a di-valent antibody fragment having a dimer Fab fragment, comprising the steps of: (a) culturing the host cell; (b) expressing the Fab fragment in the host cell; And (c) it provides a method for producing a divalent (di-valent) antibody fragment having a dimeric Fab fragment comprising the step of recombining the Fab fragment.

The Fab fragment according to the present invention specifically binds to Her2, and the two Fab fragments can bind through the SARAH domain, can be prepared homogeneously through E. coli, and the binding affinity for Her2 is Herceptin (trastuzumab ), and is useful for diagnosis, prevention and treatment of breast cancer.

1 is a schematic diagram of an antibody fragment of the present invention.
2 is a construct for each antibody fragment domain of the present invention.
3 is a schematic diagram of the construct of a re-constitutional antibody fragment of the present invention.
4 is a result of the expression pattern of antibody fragments in E. coli.
5 is a result of purification by refolding of antibody fragments.
6 is a process of purification through recombination of antibody fragments.
7 is a result of confirming the homogeneity of the antibody fragment.
FIG. 8 is a result of comparing the binding affinity of the control drug Herceptin (trastuzumab) to the antigen (Her2) of the antibody fragment.
9 is a result of comparing the binding affinity of the control drug Herceptin (trastuzumab) to the intracellular antigen (Her2) of the antibody fragment.

The present invention provides a Fab fragment that specifically binds to Her2, comprising: (a) a heavy chain variable region (V _H ) comprising an amino acid sequence represented by SEQ ID NO: 5; (b) a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 6 (C _H1 ); (c) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 11 (V _L ); And (d) a light chain constant region (C _L ) comprising an amino acid sequence represented by SEQ ID NO: 12, and a linker and a SARAH domain are further included at the C-terminus of the heavy chain constant region (C _H1 ). Characterized Fab fragments.

"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 digestion of the entire 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 the above 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 Fab fragment containing a light chain composed of the above V _L and C _L.

The Fab fragment of the present invention may contain a variant of the amino acid sequence described in the attached sequence list 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 Fab fragment specifically binding to Her2 of the present invention is a heavy chain and a light chain heterodimer. In order to form a di-valent antibody, a linker and a SARAH domain may be further linked to the C-terminus of C _H1 .

The linker may include an amino acid sequence represented by SEQ ID NO: 15.

The SARAH domain may include an amino acid sequence represented by SEQ ID NO: 16.

In addition, the present invention relates to a divalent antibody fragment comprising two or more Fab fragments, wherein the Fab fragment is bound through hydrophobic bonds between SARAH domains. By the SARAH domain, the two Fab fragments form a polymer in the opposite direction to each other, do not insert the CDK of the C _H1 figure, and insert a linker (GGGS) and SARAH domain at the ends thereof, thereby exerting the influence 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 forming a dimer through head-to-tail interaction. The dimer interface is stabilized through hydrophobic interactions.

In addition, the present invention is a Fab (Fragment antigen-binding) fragment expression cassette that specifically binds to Her2, (a) a heavy chain variable region (V _H )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 8; (b) a heavy chain constant region 1 (C _H1 )-encoding nucleic acid molecule comprising the nucleotide sequence represented by SEQ ID NO: 9; (c) a light chain variable region (V _L )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 13; And (d) a light chain constant region (C _L )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 14; Including, the heavy chain constant region 1 (C _H1 ) _-a linker to the 3'end of the coding nucleic acid molecule It relates to a fragment antigen-binding (Fab) fragment expression cassette, characterized in that the -coding nucleic acid molecule and the SARAH domain-encoding nucleic acid molecule are further included.

The linker-encoding nucleic acid molecule may include a nucleotide sequence represented by SEQ ID NO: 17.

The SARAH domain-encoding nucleic acid molecule may include a nucleotide sequence represented by SEQ ID NO: 18.

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 (Scheit, Nucleotide Analogs, John Wiley, NewYork (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 showing substantial identity to the nucleotide sequence described above. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. Of homology, more preferably at least 90% of homology, and most preferably at least 95% of homology.

The expression cassette produced in the present invention is constructed so that a desired gene can be expressed in a host cell. In general, promoters and terminators operably linked to upstream and downstream of the expression cassette 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 regulatory factor binding sites) and another nucleic acid sequence, whereby the The regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

The present invention relates to a recombinant vector comprising the expression cassette.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). 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 a 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-facing promoter of phage λ (pL λ promoter, Herskowitz) , I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as a regulatory site.

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.).

The present invention relates to a host cell transformed with the recombinant vector.

Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell can be used. For example, E. coli C43 (DE3), E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus subtilis, Bacillus thuringensis strains, and Salmonella typhimurium, Enterobacteriaceae and strains such as Serratia Marsessons and various Pseudomonas species.

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. 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 an electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)).

The present invention relates to an anticancer pharmaceutical composition comprising the Fab fragment or the bivalent antibody.

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 stomach cancer.

When the 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 in 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 is preferably applied by parenteral administration.

A suitable dosage of the pharmaceutical composition of the present invention may be variously prescribed 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. 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 unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be made by incorporating it into a multi-dose 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 detail with reference to examples and drawings. However, these examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

<Example>

1.Her2 target antibody fragment construct construction

In order to develop Her2-positive breast cancer treatment and diagnostic agents, a construct of Fab, which is an antibody fragment, was produced, and a total of four candidate substances were produced in E. coli, and the DNA sequence was determined based on the amino acid sequence and requested to Cosmo Genetech. After synthesizing the nucleotide sequence, cloning was performed based on the nucleotide sequence to prepare four candidate substances.

1) APm201, APm202 construct (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 bonds) stabilize 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, but when the hinge domain is deleted Fab fragments are formed and monovalent Fabs can be produced. In addition, the EC of the C _L domain of the Fab fragment and the cysteine of the CDK of the C _H1 domain may be linked to play an important role in forming Fab. However, cysteine can interfere with the specific site binding of the drug and diagnostic agent in the binding of maleimide cysteine in order to produce a therapeutic agent and a diagnostic agent combined with a drug and a diagnostic agent. Was prepared (see Fig. 1, Table 3 and Table 4). These, cysteine-removed V _H +C _H1 and V _L +C _L are produced as inclusion bodies in the cytoplasm when produced in Escherichia coli, and each domain is injected into one cell and expressed so that a large amount of protein can be obtained. I did.

(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 VH + 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 5 indicate variable regions. Using this, PCR primers were prepared in the regions corresponding to the VH+C _H1 and V _L +C _L domains (see Table 6), and the genes were cloned to express VH+C _H1 and V _L +C _L , respectively, and limited. Enzymatic treatment was performed and cloned into pET21-a vector (Novagen), an E. coli expression vector.

<Amino acid sequence and base sequence of Trastuzumab>

<APm201 amino acid sequence and base sequence>

<APm202 amino acid sequence and base sequence>

<APm203 amino acid sequence and base sequence>

<APm204 amino acid sequence and base sequence>

2) APm203, APm204 construct (APm203: V _H , C _H1 +SARAH+6His, APm204: V _L , C _L +GGGS+SARAH)

(1) APm203 is a V _H -C _H1 (heavy chain constant region 1: heavy chain constant region 1) of the antibody constituent domains by inserting the SARAH domain, which is a duplex domain at the C-terminus, into the intra-chain disulfide bonds. It is a di-valent Fab construct that avoids variable dimerization and secures stability by making a stable homodimer and does not inhibit the binding ability to antigens with each other by duplexing in the reverse direction. In order to form a reverse polymer by the SARAH domain, the CDK of the C _H1 domain was not inserted, GGGS (4 amino acids) was inserted at the end, and the SARAH domain was then inserted to minimize the effect of antigen binding on folding. (See Table 4 and Table 5).

(2) In addition, in the case of APm204, the EC of the V _L -C _L domain is removed to remove the disulfide bond site, and after the C _L domain, GGGS (4 amino acids) is inserted, and a SARAH domain is inserted after that to a reverse homodimer. (homodimer, see Table 5) was formed. Thereby, a construct was prepared by conjugating a drug and a diagnostic agent to remove a residue capable of forming a non-specific site bond in the binding reaction between maleimide and cysteine to produce a therapeutic agent and a diagnostic agent. In order to clone the construct, it was converted into a codon that can be expressed by E. coli, and the DNA base sequence (see Table 5) was requested to Cosmo Genetech for synthesis, and a PCR primer for the site was prepared as a template (see Table 6). ), and V _H +C _H1 -SARAH and V _L +C _L -SARAH into which the SARAH domain was inserted were cloned so that they were expressed as inclusion bodies in the cytoplasm when produced in E. coli, and each construct was 1 By injecting into dog cells and expressing them, a large amount of protein could be obtained.

Construct Primer Sequence (5' → 3') APm201_F(Nde1): ggaattcCATATGGAAGTTCAGCTGGTTGAATC APm201_R(Xho1): ccgCTCGAGGCTTTTCGGCG APm202_F(Nde1) ggaattcCATATGGATATTCAGATGACCCAGAGCC APm202_R(stop-Xho1) ccgCTCGAGCTAGCCGCGGTTAAAGC APm203_Fd_F(Nde1) ggaattcCATATGGAAGTTCAGCTGGTTGAATC APm203_Fd_R(BamH1) cgGGATCCGCCACCGCTTTT CGGCGGC APm203_Sarah_F(BamH1) cgGGATCCGATTTTGACTTCCTGAAAAACC APm203_Sarah_R(Xho1) ccgCTCGAGTTTAGCGTCCATCG APm204_Lc_F(Nde1) ggaattcCATATGGATATTCAGATGACCCAGAGCC APm204_Lc_R(BamH1) cgGGATCCACCGCCGCGGTTAAAGC APm204_Sarah_F(BamH1) cgGGATCCGATTTTGACTTCCTGAAAAACC APm204_Sarah_R(stop-Xho1) ccgCTCGAGCTATTTAGCGTCCATCG

<Primer sequence for cloning antibody fragments>

2. Confirmation of E. coli expression of each construct

1) Confirmation of E. coli expression of APm201 and APm202 constructs

In order to confirm the expression of the genes of the APm201 and APm202 constructs, BL21 (DE3, pLysS) cells, which are E. coli-expressing cell lines, were transformed at 42°C through heat shock, and then one colony in LB medium (single colony). ) Was inoculated and cultured at 37° C. for 7 hours, treated with 0.5 mM IPTG, and cultured at 15° C. and 37° C. for 4 hours, and then APm201 and APm202 expressions were confirmed through 12% SDS-PAGE (see FIG. 4).

As a result of expression of APm201 and APm202 cultured under the same culture conditions, it was confirmed that the expression levels of APm201 and APm202 were higher at 37°C than at 15°C, and were not expressed in a soluble state, but were expressed in the form of inclusion bodies, respectively.

2) Confirmation of E. coli expression of APm203 and APm204 constructs

In order to confirm the expression of the genes of the APm203 and APm204 constructs, BL21 (DE3, pLysS) cells, which are E. coli-expressing cell lines, were transformed through heat shock at 42°C, followed by one single colony in LB medium. ) Was inoculated and cultured at 37° C. for 7 hours, treated with 0.5 mM IPTG, and cultured at 15° C. and 37° C. for 4 hours, and then APm203 and APm204 expressions were confirmed through 12% SDS-PAGE.

As a result of the expression of APm203 and APm204 cultured under the same culture conditions, the expression pattern of APm201 and APm202 was the same as that of APm201 and APm202, and the amount of expression was higher at 37°C than at 15°C. It was confirmed that it was expressed.

3. Culture and purification of antibody fragments

1) Culture and purification of APm201 and APm202 antibody fragments

The host cells for the purification of the antibody fragment were E. coli BL21 (DE3, pLysS) for recombinant protein overexpressing T7 polymerase, 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 a single colony is inoculated into LB medium containing ampicillin at 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, when the OD ₆₀₀ value reaches between 1 and 2, 0.2 mM IPTG was treated and incubated at 37° C. 150 rpm for 5 hours. 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 culture medium was added to the cells expressing the antibody fragment and mixed with the cells. Thereafter, the cells were lysed for 10 minutes (pulse on 1 second, pulse off 1 second) using an ultrasonic disruptor. Centrifugation was performed for 40 minutes at 4° C. and 10,000 rpm using a centrifuge to separate the antibody fragments, proteins and cells on the inclusion body.

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 of culture medium and obtain a pellet again. Centrifugation was performed for 40 minutes at 4° C. and 10,000 rpm using a centrifuge. In order to melt the inclusion body containing the antibody fragment thus obtained, it was melted using a melting buffer [8M urea pH 8.0] per 1 liter of cells, and then stabilized by incubating at 55°C for 40 minutes. In order to remove the material, centrifugation was performed at 4° C. and a speed of 10,000 rpm for 40 minutes. Antibody fragments melted in 8M urea were obtained and used, APm202 was used for recombination with the V _H -C _H1 domain in a molten state in 8M urea, and APm201 was used for recombination with V _L -C _L after purification. (See Fig. 5).

By filling the APm201 aqueous solution melted in 8M urea into an open column with NI-NTA resin that can bind to 6his at the C-terminus of APm201, equilibrating with 5CV (5 times column volume) of 8M urea pH 8.0 and then flowing it. After allowing the resin and antibody fragments to bind, 5CV (5 times column volume) of 8M urea washing buffer (8M urea pH 8.0) was poured, and then 5CV of 4M urea washing buffer [50 mM Tris pH 7.5, 500 mM NaCl , 4M urea, 70 mM imidazole] to remove nonspecifically bound impurities and to 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, 4M urea, 500 mM imidazole] was flowed. As can be seen from the experimental results, as a result of expressing the APm201 and APm202 constructs in the pET21 vector, it was confirmed that the antibody fragment was expressed in the form of an inclusion body.

2) Culture and purification of APm203 and APm204 antibody fragments

The host cells for the purification of the antibody fragment were E. coli BL21 (DE3, pLysS) 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. ) To produce antibody fragments.

The medium used for seed culture to produce each protein is Lysogeny broth (LB). One colony was inoculated into LB medium containing ampicillin and cultured overnight at 150 rpm using a shaking incubator at 37°C. I did. Then, inoculate the cell culture medium in 1L TB medium (Gellix) containing new ampicillin with approximately 0.5 to 2% of the cell culture medium and incubate with shaking at 150 rpm for 5 hours, and then treat 0.2 mM IPTG when the OD ₆₀₀ value reaches between 1 and 2 Then, it was 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 at -80°C and used. 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 culture medium was added to the cells expressing the antibody fragment and mixed 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 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% TritonX-100] per 1 L of culture medium and centrifuge to obtain a pellet again. Centrifugation was performed for 40 minutes at 4° C. and 10,000 rpm using a separator. In order to melt the inclusion body containing the antibody fragment thus obtained, melt it using a melting buffer [8M urea pH 8.0] per 1 liter of cells, incubate at 55°C for 40 minutes to stabilize, and then remove the unmelted material again. In order to do so, centrifugation was performed for 40 minutes at a speed of 4° C. and 10,000 rpm. The antibody fragments melted in 8M urea were obtained, and APm203 and APm204 were purified and used for recombination (see FIG. 5).

The APm203 and APm204 aqueous solutions melted in 8M urea were filled with NI-NTA resin capable of binding to 6his at the C-terminus of APm203 and APm204 in an open column, and equilibrated with 5CV (5 times the column volume) of 8M urea pH 8.0. After this, the resin and antibody fragments are allowed to bind by flowing, and then 5CV (5 times column volume) of washing buffer (8M urea pH 8.0) is poured, and then 5CV of 4M urea washing buffer [50 mM Tris, pH 7.5, 500 mM NaCl, 4M urea, 70 mM imidazole] was added to remove non-specifically bound impurities and lower the urea concentration. To separate the APm203 and APm204 antibody fragments from the Ni-NTA resin, an elution buffer [elution buffer; 50 mM Tris pH 7.5, 500 mM NaCl, 4M urea, 500 mM imidazole] was flowed (see FIG. 5). As can be seen from the experimental results, as a result of expressing the APm203 and APm204 constructs in the pET21 vector, the antibody fragment was expressed in the form of an inclusion body, and it was confirmed that they exist in a molten form in 8M urea (see FIG. 5). ).

3) 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 suspension with [50 mM Tris, pH 7.5, 500 mM NaCl, 10% glycerol], 100 at 4° C. for 3 days to induce hydrophobic binding between APm201 (V _H +CH1) and APm202 (V _L +C _L ). React on 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 portion to produce APm208 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 equilibrating with 5CV (column volume) and flowing to allow the resin and APm201 to bind, 5CV (column volume) washing buffer [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 70 mM imidazole] was flowed to remove APm202 that 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 (see FIG. 6). As can be seen from the experimental results, it was confirmed that APm208 recombined through hydrophobic bonding in the solutions of APm201 and APm202.

4) Production of APd209 through recombination of APm202 and APm203

For recombination of the antibody fragments melted in urea, APm202 (V _L +C _L ) and APm203 (V _H +C _H1 +SARAH domain) were mixed in a ratio of 1: 3 so that the final urea concentration was 0.5 M or less. To induce hydrophobic binding of APm202 (V _L +C _L ) and APm203 (V _H +C _H1 +SARAH domain) after suspending in a dilution buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol] It is reacted at 100 rpm at 4° C. for 3 days.

In order to remove the precipitate of the solution reacted with APm202 (V _L +C _L ) and APm203 (V _H +C _H1 +SARAH domain), 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 APd209 in the aqueous phase, NI-NTA resin that can bind to 6his at the C-terminus of APm203 (V _H +C _H1 +SARAH domain) was filled, and a buffer solution [50 After equilibrating with 5CV (column volume) of mM Tris pH 7.5, 500 mM NaCl, 10% glycerol], the resin and APm203 are allowed to bind by flowing, and then 5CV (column volume) washing buffer [50 mM Tris pH 7.5] , 500 mM NaCl, 10% glycerol, 70 mM imidazole] to remove APm202, which did not bind to APm203. In order to separate the antibody fragments of APm202 and APd209 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 (see FIG. 6). As can be seen from the experimental results, it was confirmed that APd209 recombined through hydrophobic bonding in the solutions of APm202 and APm203.

5) Production of APd210 through recombination of APm201 and APm204

For recombination of the antibody fragments melted in urea, APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAH domain) were each mixed at a ratio of 1:3 and the final urea concentration was 0.5 M or less. After suspending in a dilution buffer solution [50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol], for 3 days to induce hydrophobic binding between APm201 (V _H +C _H1 ) and APm202 (V _L +C _L ). It is reacted at 4° C. at 100 rpm. In order to remove the precipitate of the solution reacted with APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAH domain), 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 APd210 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 equilibrating with 5CV (column volume) and flowing to allow the resin and APm201 to bind, 5CV (column volume) washing buffer [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 APd210 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 (see FIG. 9). As can be seen from the experimental results, it was confirmed that APd210 was recombined through hydrophobic bonding in the solutions of APm201 (V _H +C _H1 ) and APm204 (V _L +C _L +SARAHdomain).

4. Confirmation of homogeneity of APm208, APd209 and APd210

Size exclusion chromatography was performed to confirm the homogeneity of the refold and recombinant antibody fragments APm208, and APd209. 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. The antibody fragment aqueous solution eluted during the recombination process was flowed onto the column, and the retention volume was measured to be 70-72 ml. From the 280 nm absorbance indicating the antibody fragment in the chromatogram and the results of the SDS-PAGE experiment, the homogeneity of the antibody fragment is considered to be very high (see Fig. 7).

5. sHer2 binding affinity measurement using enzyme-linked immunosorbent assay (ELISA)

Add 100 ng/well of antigen Her2 (extracellular domain) to a 96-well ELISA plate, react overnight at 4° C. to immobilize the antigen on the plate surface, remove the supernatant, and add 200 μl of a blocking solution (Sigma, B6429-500ML). 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 to a concentration of 0-125 ng/ml using PBS. Each 100 µl of each was dispensed and reacted for 1 hour at room temperature 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, dispensed into each well by 100 µl, and reacted for 1 hour at room temperature, and then the supernatant was removed and washed three 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 for 1 hour at room temperature. 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 absorbance of 450 nm wavelength was measured using a microplate reader. The binding affinity compared to trastuzumab was measured to be APm208 (61.51%), APd209 (160.2%), and APd210 (79.92%) (see Fig. 8), which means that the activity of the antibody fragment against Her2 is sufficiently maintained.

6. sHer2-binding affinity measurement using fluorescence-activated cell sorting (FACS)

SKOV3, a cell line overexpressing Her2, was cultured, and 4 × 10 ⁶ cells were suspended in 2 ml of FACS buffer (PBS, 2% FBS), and 50 μl of each well of a 96-well plate was dispensed. Trastuzumab as a control group and purified antibody fragments as an experimental group were diluted with PBS to a concentration of 0-200 nM, and then 50 µl was dispensed into each well of the plate where cells were dispensed, and mixed well with cells. After locking, it is reacted at 4℃ for 20 minutes. After the reaction, centrifugation at 4° C. at 300 × g and the supernatant is removed. The precipitated cells were suspended in 200 µl of PBS, centrifuged at 300 × g at 4° C., and the washing process of removing the supernatant was repeated twice. Secondary antibodies (sigma, F5512) were diluted 1/32 in PBS, dispensed and suspended in each well by 100 μl, and reacted at 4° C. for 20 minutes. After the reaction, the mixture was centrifuged at 4° C. at 300 × g and the supernatant was removed. After the cells of each well were suspended in 200 µl of PBS, the cells were centrifuged at 4° C. at 300 × g, and the washing process of removing the supernatant was repeated twice. Thereafter, the cells were suspended in 300 µl of FACS buffer, and the number of cells bound to the secondary antibody was counted using a Flow Cytometer (BD FACSCalibur), and compared with the control group, trastuzumab. The binding affinity compared to trastuzumab was measured to be APm208 (84.9%), APd209 (89.5%), and APd210 (90.8%), which means that the activity of the antibody fragment against Her2 is sufficiently maintained (see Fig. 9).

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Novel di-valent antibody fragment platform <130> P190126 <160> 18 <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> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 15 Gly Gly Gly Ser One <210> 16 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> SARAH <400> 16 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> 17 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Linker <400> 17 ggtggcggat cc 12 <210> 18 <211> 147 <212> DNA <213> Artificial Sequence <220> <223> SARAH <400> 18 gattttgact tcctgaaaaa cctgagttta gaagaactgc agatgcgtct gaaagcactg 60 gacccgatga tggaacgtga aatcgaagag ctgcgtcagc gttacaccgc caaacgtcag 120 ccgattctgg acgcgatgga cgctaaa 147

Claims (12)

As a di-valent antibody fragment having a dimer Fab fragment, the Fab fragment
(a) a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 5 (V _H );
(b) a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 6 (C _H1 );
(c) a light chain variable region (V _L ) comprising an amino acid sequence represented by SEQ ID NO: 11; And
(d) comprising a light chain constant region (C _L ) comprising the amino acid sequence represented by SEQ ID NO: 12,
The heavy chain constant region (C _H1 ) is a di-valent (di-valent) antibody fragment, characterized in that further comprising a linker and a SARAH domain at the C-terminus of the heavy chain constant region (C _H1 ). The di-valent antibody fragment according to claim 1, wherein the linker comprises an amino acid sequence represented by SEQ ID NO: 15. The di-valent antibody fragment according to claim 1, wherein the SARAH domain comprises an amino acid sequence represented by SEQ ID NO: 16. The di-valent antibody fragment of claim 1, wherein the Fab fragment is recombined through hydrophobic bonds between SARAH domains. As a Fab fragment expression cassette for a di-valent antibody fragment having a dimer Fab fragment,
(a) a heavy chain variable region (V _H )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 8;
(b) a heavy chain constant region 1 (C _H1 )-encoding nucleic acid molecule comprising the nucleotide sequence represented by SEQ ID NO: 9;
(c) a light chain variable region (V _L )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 13; And
(d) a light chain constant region (C _L )-encoding nucleic acid molecule comprising a nucleotide sequence represented by SEQ ID NO: 14; Including,
Fab fragment expression cassette, characterized in that the linker-encoding nucleic acid molecule and the SARAH domain-encoding nucleic acid molecule are further included at the 3'end of the heavy chain constant region 1 (C _H1 )-encoding nucleic acid molecule. The Fab fragment expression cassette according to claim 5, wherein the linker-encoding nucleic acid molecule comprises a nucleotide sequence represented by SEQ ID NO: 17. The Fab fragment expression cassette according to claim 5, wherein the SARAH domain-encoding nucleic acid molecule comprises a nucleotide sequence represented by SEQ ID NO: 18. A recombinant vector comprising the expression cassette of claim 5. A host cell transformed with the recombinant vector of claim 8. A pharmaceutical composition for anticancer, characterized in that it comprises the divalent (di-valent) antibody fragment of claim 1. 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 liver cancer, lung cancer, head and neck cancer, esophageal cancer and stomach cancer. As a method for preparing a di-valent antibody fragment having a dimer Fab fragment,
(a) culturing the host cell of claim 9;
(b) expressing the Fab fragment in the host cell; And
(c) recombining the Fab fragment
Method for producing a divalent (di-valent) antibody fragment having a dimer Fab fragment, characterized in that it comprises a.

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